Monoclonal antibody hPAM4

ABSTRACT

This invention relates to monovalent and multivalent, monospecific antibodies and to multivalent, multispecific antibodies. One embodiment of these antibodies has one or more identical binding sites where each binding site binds with a target antigen or an epitope on a target antigen. Another embodiment of these antibodies has two or more binding sites where these binding sites have affinity towards different epitopes on a target antigen or different target antigens, or have affinity towards a target antigen and a hapten. The present invention further relates to recombinant vectors useful for the expression of these functional antibodies in a host. More specifically, the present invention relates to the tumor-body associated antibody designated PAM4. The invention further relates to humanized and human PAM4 antibodies, and the use of such antibodies in diagnosis and therapy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/388,314, filed Jun. 14, 2002, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to monovalent and multivalent, monospecificantibodies and to multivalent, multispecific antibodies. Specifically,the present invention relates to a MUC1 antigen specific antibodydesignated PAM4. The invention further relates to humanized and humanPAM4 antibodies and fragments thereof, and the use of such antibodiesand fragments thereof in diagnosis and therapy.

In one embodiment, the antibodies of the present invention have one ormore identical binding sites, wherein each binding site has an affinitytoward a target antigen or an epitope on a target antigen. In anotherembodiment, the antibodies of the present invention have two or morebinding sites which have an affinity toward the same or differentepitopes on a target antigen or the same or different target antigens,or at least one binding site has an affinity toward a target antigen andat least one binding site has an affinity toward a hapten. The presentinvention also describes recombinant vectors useful for expressing theantibodies described herein in a host.

BACKGROUND OF THE INVENTION

The pancreas produces insulin to assist the body in converting glucoseto energy and enzymes to assist the body in digesting food. Pancreaticcancer is a malignant growth of the pancreas that mainly occurs in thecells of the pancreatic ducts. This disease is the ninth most commonform of cancer, yet it is the fourth and fifth leading cause of cancerdeaths in men and women, respectively. Cancer of the pancreas is almostalways fatal, with a five-year survival rate that is less than 3%.

The most common symptoms of pancreatic cancer include jaundice,abdominal pain, and weight loss, which, together with other presentingfactors, are nonspecific in nature. Thus, diagnosing pancreatic cancerat an early stage of tumor growth is often difficult and requiresconsiderable suspicion and extensive diagnostic work-up, often timesincluding exploratory surgery. Endoscopic ultrasonography and computedtomography are the best noninvasive means available today for diagnosisof pancreatic cancer. However, reliable detection of small tumors, aswell as differentiation of pancreatic cancer from focal pancreatitis, istroublesome. Unfortunately, the vast majority of patients are presentlydiagnosed at a late stage when the tumor has already extended outside ofthe capsule to invade surrounding organs and/or has metastasizedextensively. Gold et al., Crit. Rev. Oncology/Hematology, 39:147-54(2001). Late detection of the disease is common, and “early” pancreaticcancer diagnosis is rare in the clinical setting.

Current treatment procedures available for pancreatic cancer have notled to a cure, nor to a substantially improved survival time. Surgicalresection has been the only modality that offers a chance at survival.However, due to a large tumor burden, only 10% to 25% of patients arecandidates for “curative resection.” For those patients undergoing asurgical treatment, the five-year survival rate is still poor, averagingonly about 10%.

Early detection and diagnosis of pancreatic cancer, as well asappropriate staging of the disease, would provide an increased survivaladvantage. A number of laboratories are proceeding on the development ofa diagnostic procedure based upon the release of a tumor-associatedmarker into the bloodstream as well as detection of the marker substancewithin biopsy specimens. The best tumor associated marker for pancreaticcancer has been the immunoassay for CA19.9. Elevated levels of thissialylated Lea epitope structure were found in 70% of pancreatic cancerpatients but were not found in any of the focal pancreatitis specimensexamined. However, CA19.9 levels were found to be elevated in a numberof other malignant and benign conditions, so that currently the assaycannot be used for diagnosis. However, the assay is useful formonitoring, the continued increase in CA19.9 serum levels after surgerybeing indicative of a poor prognosis. Many other monoclonal antibodies(MAbs) have been reported with immunoassays for diagnosis in varyingstages of development. These include but are not limited to DUPAN2,SPAN1, B72.3, Ia3, and various anti-CEA antibodies.

Man-made antibodies, in particular MAbs and engineered antibodies orantibody fragments, have been tested widely and shown to be of value indetection and treatment of pancreatic cancer, as well as other varioushuman disorders, including cancers, autoimmune diseases, infectiousdiseases, inflammatory diseases, and cardiovascular diseases [Filpulaand McGuire, Exp. Opin. Ther. Patents (1999) 9: 231-245]. The clinicalutility of an antibody or an antibody-derived agent is primarilydependent on its ability to bind to a specific targeted antigenassociated with a specific disorder. Selectivity is valuable fordelivering a diagnostic or therapeutic agent, such as isotopes, drugs,toxins, cytokines, hormones, hormone antagonists, enzymes, enzymeinhibitors, oligonucleotides, growth factors, oligonucleotides,radionuclides, an angiogenesis inhibitor, or metals, to a targetlocation during the detection and treatment phases of a human disorder,particularly if the diagnostic or therapeutic agent is toxic to normaltissue in the body. Radiolabeled antibodies have been used with somesuccess in numerous malignancies, including ovarian cancer, colon cancerand lymphoma. This technology may also prove useful for pancreaticcancer. However, other than the application of anti-CEA antibodies andB72.3, little clinical information exists.

The potential limitations of such antibody systems are discussed inGoldenberg, The American Journal of Medicine, 94: 298-299 (1993). Theimportant parameters in the detection and treatment techniques are theamount of the injected dose specifically localized at the site(s) wheretarget cells are present and the uptake ratio, i.e. the ratio of theconcentration of specifically bound antibody to that of theradioactivity present in surrounding normal tissues. When an antibody isinjected into the blood stream, it passes through a number ofcompartments as it is metabolized and excreted. The antibody must beable to locate and bind to the target cell antigen while passing throughthe rest of the body. Factors that control antigen targeting includelocation, size, antigen density, antigen accessibility, cellularcomposition of pathologic tissue, and the pharmacokinetics of thetargeting antibodies. Other factors that specifically affect tumortargeting by antibodies include expression of the target antigens, bothin tumor and other tissues, and bone marrow toxicity resulting from theslow blood-clearance of the radiolabeled antibodies. The amount oftargeting antibodies accreted by the targeted tumor cells is influencedby the vascularization and barriers to antibody penetration of tumors,as well as intratumoral pressure. Non-specific uptake by non-targetorgans such as the liver, kidneys or bone-marrow is another potentiallimitation of the technique, especially for radioimmunotherapy, whereirradiation of the bone marrow often causes the dose-limiting toxicity.

One suggested approach for delivering agents to a target site, referredto as direct targeting, is a technique designed to target specificantigens with antibodies carrying diagnostic or therapeuticradioisotopes. In the context of tumors, the direct targeting approachutilizes a radiolabeled anti-tumor monospecific antibody that recognizesthe target tumor through its antigens. The technique involves injectingthe labeled monospecific antibody into the patient and allowing theantibody to localize at the target tumor to obtain diagnostic ortherapeutic benefits. The unbound antibody clears the body. Thisapproach can be used to diagnose or treat additional mammaliandisorders.

Another suggested solution, referred to as the “Affinity EnhancementSystem” (AES), is a technique especially designed to overcomedeficiencies of tumor targeting by antibodies carrying diagnostic ortherapeutic radioisotopes [U.S. Pat. No. 5,256,395 (1993), Barbet etal., Cancer Biotherapy & Radiopharmaceuticals 14: 153-166 (1999)]. TheAES utilizes a radiolabeled divalent hapten and ananti-tumor/anti-hapten bispecific antibody that recognizes both thetarget tumor and the radioactive hapten. Haptens with higher valency andantibodies with higher specificity may also be utilized for thisprocedure. The technique involves injecting the antibody into thepatient and allowing it to localize at the target tumor. After asufficient amount of time for the unbound antibody to clear from theblood stream, the radiolabeled hapten is administered. The hapten bindsto the antibody-antigen complex located at the site of the target cellto obtain diagnostic or therapeutic benefits, while the unbound haptenrapidly clears from the body. Barbet mentions the possibility that abivalent hapten may crosslink with a bispecific antibody, when thelatter is bound to the tumor surface. As a result, the radiolabeledcomplex is more stable and stays at the tumor for a longer period oftime. This system can be used to diagnose or treat mammalian disorders.

There remains a need in the art for production of multivalent,monospecific antibodies that are useful in a direct targeting system andfor production of multivalent, multispecific antibodies that are usefulin an affinity enhancement system. Specifically, there remains a needfor a antibody that performs as a useful diagnostic tool for pancreaticcancer and that exhibits enhanced uptake at targeted antigens, decreasedconcentration in the blood, and optimal protection of normal tissues andcells from toxic pharmaceuticals.

SUMMARY OF THE INVENTION

Contemplated in the present invention is an antibody, fusion protein,and fragments thereof that bind a domain located between the aminoterminus and start of the repeat domain of MUC1. In a preferredembodiment the antibody, fusion protein, or fragment thereof is a PAM4antibody. The PAM4 antibody, fusion protein, or fragment thereof of thepresent invention is derived by immunization and/or selection withmucin, and is preferably reactive against mucin of pancreatic cancer.Accordingly, the PAM4 antibody, fusion protein, and fragments thereof ofthe present invention preferably bind an antigen associated withpancreatic cancer cells.

In a preferred embodiment, the PAM4 antibody or fragment thereof ishumanized or fully human, or the PAM4 fusion protein comprises ahumanized or fully human PAM4 antibody or fragment thereof. Alsopreferred, the PAM4 antibody, fusion protein, and fragments thereof canbe conjugated to at least one therapeutic and/or diagnostic agent.

Contemplated herein is a humanized PAM4 antibody or fragment thereofcomprising the complementarity-determining regions (CDRs) of a murinePAM4 MAb and the framework (FR) regions of the light and heavy chainvariable regions of a human antibody and the light and heavy chainconstant regions of a human antibody, wherein the CDRs of the lightchain variable region of the humanized PAM4 MAb comprise CDR1 comprisingan amino acid sequence of SASSSVSSSYLY; CDR2 comprising an amino acidsequence of STSNLAS; and CDR3 comprising an amino acid sequence ofHQWNRYPYT; and the CDRs of the heavy chain variable region of thehumanized PAM4 MAb comprise CDR1 comprising an amino acid sequence ofSYVLH; CDR2 comprising an amino acid sequence of YINPYNDGTQYNEKFKG andCDR3 comprising an amino acid sequence of GFGGSYGFAY. In a preferredembodiment, the FRs of the light and heavy chain variable regions of thehumanized PAM4 antibody or fragment thereof comprise at least one aminoacid substituted from the corresponding FRs of a murine PAM4 MAb. Stillpreferred, the humanized PAM4 antibody or fragment thereof of comprisesat least one amino acid selected from the group consisting of amino acidresidues 5, 27, 30, 38, 48, 66, 67, and 69 of the murine heavy chainvariable region of FIG. 1B, PAM4 VH amino acid sequence. Also preferred,the humanized PAM4 antibody or fragment thereof wherein said amino acidfrom said murine MAb is at least one amino acid selected from the groupconsisting of amino acid residues 21, 47, 59, 60, 85, 87, and 100 of themurine light chain variable region FIG. 1A, PAM4Vκ sequence. Mostpreferably, the humanized PAM4 antibody or fragment thereof comprisesthe PAM4 Vκ nucleotide sequence of FIG. 1A and the PAM4 VH nucleotidesequence of FIG. 1B and/or comprises a hPAM4 VH amino acid sequence ofFIG. 4A and a hPAM4 Vκ amino acid sequence of FIG. 4B.

Another embodiment of the present invention is a cancer cell targetingdiagnostic immunoconjugate comprising an antibody component thatcomprises an antibody or fragment thereof of any one of the antibodies,fusion proteins, or fragments thereof of the present invention, whereinthe antibody, fusion protein, or fragment thereof is bound to at leastone diagnostic/detection agent.

Preferably, the diagnostic/detection agent is selected from the groupcomprising a radionuclide, a contrast agent, and a photoactivediagnostic/detection agent. Still preferred, the diagnostic/detectionagent is a radionuclide with an energy between 20 and 4,000 keV or is aradionuclide selected from the group consisting of ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu,¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc,^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P, ¹¹C, ¹³N, ¹⁵O,¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br, ^(82m)Rb, ⁸³Sr, orother gamma-, beta-, or positron-emitters. Also preferred, thediagnostic/detection agent is a paramagnetic ion, such as the a metalcomprising chromium (III), manganese (II), iron (III), iron (II), cobalt(II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III), or a radioopaquematerial, such as barium, diatrizoate, ethiodized oil, gallium citrate,iocarmic acid, iocetamic acid, iodamide, iodipamide, iodoxamic acid,iogulamide, iohexol, iopamidol, iopanoic acid, ioprocemic acid,iosefamic acid, ioseric acid, iosulamide meglumine, iosemetic acid,iotasul, iotetric acid, iothalamic acid, iotroxic acid, ioxaglic acid,ioxotrizoic acid, ipodate, meglumine, metrizamide, metrizoate,propyliodone, and thallous chloride.

Also preferred, the diagnostic/detection agent is a fluorescent labelingcompound selected from the group comprising fluorescein isothiocyanate,rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine, a chemiluminescent labeling compound selected fromthe group comprising luminol, isoluminol, an aromatic acridinium ester,an imidazole, an acridinium salt and an oxalate ester, or abioluminescent compound selected from the group comprising luciferin,luciferase and acquorin. In another embodiment, the diagnosticimmunoconjugates of the present invention are used in intraoperative,endoscopic, or intravascular tumor diagnosis.

Another embodiment of the present invention is a cancer cell targetingtherapeutic immunoconjugate comprising an antibody component thatcomprises an antibody or fragment thereof of any one of the antibodies,fusion proteins, or fragments thereof of the present invention, whereinthe antibody, fusion protein, or fragment thereof is bound to at leastone therapeutic agent.

Preferably, the therapeutic agent is selected from the group consistingof a radionuclide, an immunomodulator, a hormone, a hormone antagonist,an enzyme, oligonucleotide, an enzyme inhibitor, a photoactivetherapeutic agent, a cytotoxic agent, an angiogenesis inhibitor, and acombination thereof.

In one embodiment, an oligonucleotide, such as an antisense moleculeinhibiting bcl-2 expression is described in U.S. Pat. No. 5,734,033(Reed), which is incorporated by reference in its entirety, may beconjugated to, or form the therapeutic agent portion of animmunoconjugate or antibody fusion protein of the present invention.Alternatively, the oligonucleotide may be administered concurrently orsequentially with a naked or conjugated PAM4 antibody or antibodyfragment of the present invention. In a preferred embodiment, theoligonucleotides is an antisense oligonucleotide that preferably isdirected against an oncogene or oncogene product of a B-cell malignancy,such as bcl-2.

In a preferred embodiment, the therapeutic agent is a cytotoxic agent,such as a drug or a toxin. Also preferred, the drug is selected from thegroup consisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogs,anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purineanalogs, antibiotics, enzymes, enzyme inhibitors, epipodophyllotoxins,platinum coordination complexes, vinca alkaloids, substituted ureas,methyl hydrazine derivatives, adrenocortical suppressants, hormoneantagonists, endostatin, taxols, camptothecins, SN-38, doxorubicins andtheir analogs, antimetabolites, alkylating agents, antimitotics,antiangiogenic, apoptotoic agents, methotrexate, CPT-11, and acombination thereof.

In another embodiment, the therapeutic agent is an oligonucleotide. Forexample, the oligonucleotide may be an antisense oligonucleotide such asan antisense oligonucleotide against an oncogene like bcl-2 and p53.

In another preferred embodiment, the therapeutic agent is a toxinselected from the group consisting of ricin, abrin, alpha toxin,saporin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonasexotoxin, and Pseudomonas endotoxin and combinations thereof, animmunomodulator is selected from the group consisting of a cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), a stem cell growthfactor, erythropoietin, thrombopoietin and a combinations thereof, aradionuclide selected from the group consisting of ³²P, ³³P, ⁴⁷Sc, ⁶⁴Cu,⁶⁷Cu, ⁶⁷Ga, ⁸⁶Y, ⁹⁰Y, ¹¹¹Ag, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At,²²³Ra and ²²⁵Ac, and combinations thereof, or a photoactive therapeuticagent selected from the group comprising chromogens and dyes.

Still preferred, the therapeutic agent is an enzyme selected from thegroup comprising malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,α-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, β-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

Contemplated herein is a multivalent, multispecific antibody or fragmentthereof comprising more than one antigen binding site having an affinitytoward a PAM4 target antigen and one or more hapten binding sites havingaffinity towards hapten molecules. Preferably, the antibody or fragmentthereof is a humanized or fully human antibody or fragment thereof. Alsopreferred, the multivalent, multispecific antibody or fragment thereoffurther comprises a diagnostic/detection and/or therapeutic agent.

Also described herein is a bispecific antibody or fragment thereofcomprising at least one bnding site with an affinity toward a PAM4target antigen and at least one binding site with an affinity toward atargetable construct/conjugates selected from the group consisting of:

-   -   DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH₂ (IMP 271);    -   DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂ (IMP 277);    -   DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂ (IMP 288);    -   DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂ (IMP 0281); and    -   DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (IMP 284),        that is capable of carrying at least one diagnostic and/or        therapeutic agent. Other targetable constructs suitable for use        in the present invention are disclosed in U.S. Provisional        Application entitled “D-Amino Acid Peptides” (McBride), Attorney        Docket Number 018733/1206, filed Jun. 13, 2003.

Another embodiment of the present invention is an antibody fusionprotein or fragment thereof comprising at least two PAM4 MAbs orfragments thereof, wherein the MAbs or fragments comprise any of theantibodies and fragments thereof of the present invention. Alsopreferred, the antibody fusion protein or fragment thereof comprises atleast one first PAM4 MAb or fragment thereof of any one of theantibodies and fragments thereof of the present invention and at leastone second MAb or fragment thereof, other than the MAb or fragmentthereof of the antibodies and fragments thereof of the presentinvention. Preferably, the second MAb is a carcinoma-associatedantibody, preferably selected from the group consisting of CA19.9,DUPAN2, SPAN1, Nd2, B72.3, CC49, CEA, aLe^(a), antibodies defined by theLewis antigen Le(y), and antibodies against CSAp, insulin-like growthfactor (IGF), CD-80, placental growth factor (PlGF), carbonic anhydraseIX, tenascin, IL-6, MUC2, MUC3, MUC4, TAG-72, EGFR, CD40, plateletderived growth factor, IL-6, angiogenesis factors (e.g., VEGF), productsof oncogenes, HER2/neu and antigens associated with renal cancer,gastric cancer and melanoma. The antibody fusion protein or fragmentsthereof of the present invention may further comprises at least onediagnostic and/or therapeutic agent.

Also described herein is a DNA sequence comprising a nucleic acidencoding a MAb or fragment thereof selected from the group consistingof:

-   -   (a) a PAM4 antibody or fragment thereof of any one of the        antibodies described in the present invention;    -   (b) an antibody fusion protein or fragment thereof comprising at        least two of the MAbs or fragments thereof described in (a);    -   (c) an antibody fusion protein or fragment thereof comprising at        least one first PAM4 MAb or fragment thereof comprising said MAb        or fragment thereof of the PAM4 antibodies or fragments thereof        of the present invention and at least one second MAb or fragment        thereof, other than the MAb or fragment thereof of any one of        the antibodies or fragments thereof of the present invention;        and    -   (d) an antibody fusion protein or fragment thereof comprising at        least one first MAb or fragment thereof comprising said MAb or        fragment thereof of any one of the antibodies or fragments        thereof of the present invention and at least one second MAb or        fragment thereof, other than the MAb or fragment thereof of any        one of antibodies or fragments thereof of the present invention,        wherein the second MAb is a carcinoma associated antibody.        Preferably, the carcinoma associated antibody is selected from        the group consisting of CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49,        CEA, aLe^(a), antibodies defined by the Lewis antigen Le(y), and        antibodies against CD40, CD80, angiogenesis factors (e.g., VEGF        and PlGF), carbonic anhydrase IX, products of oncogenes, MUC1,        MUC-2, MUC-3, MUC-4, TAG-72, EGFR, insulin-like growth factor        (IGF), platelet derived growth factor, tenascin, CD30, CD33,        HLA-DR, IL-6, HER2/neu and antigens associated with renal        cancer, gastric cancer and melanoma.

Also described in the present invention is an expression vector and hostcell comprising the DNA sequence of any one of the antibodies, fusionproteins or fragments thereof of the present invention.

Another embodiment of the present invention is a method of delivering adiagnostic or therapeutic agent, or a combination thereof, to a targetcomprising (i) providing a composition that comprises a PAM4 antibody orfragment thereof conjugated to at least one diagnostic/detection and/ortherapeutic agent and (ii) administering to a subject in need thereofthe diagnostic or therapeutic conjugate of any one of antibodies, fusionproteins, or fragments thereof of the present invention. Preferably, thediagnostic/detection agent is selected from the group consisting of aradionuclide, a contrast agent, and a photoactive diagnostic/detectionagent, and the therapeutic agent is preferably selected from the groupconsisting of a drug, toxin, cytotoxic agent, cytokine, immunomodulator,hormone, hormone antagonist, growth factor, radionuclide, metal.

Also contemplated in the present invention is a method of delivering adiagnostic/detection agent, a therapeutic agent, or a combinationthereof to a target, comprising: (a) administering to a subject theantibody or fragment thereof of any one of the multivalent,multispecific antibodies or fragments thereof of the present inventionthat have an affinity toward a PAM4 antigen and comprise one or morehapten binding site; (b) waiting a sufficient amount of time for anamount of the non-antibody to clear the subject's blood stream; and (c)administering to said subject a carrier molecule comprising adiagnostic/detection agent, a therapeutic agent, or a combinationthereof, that binds to a binding site of the antibody. Preferably, thecarrier molecule binds to more than one binding site of the antibody.Still preferred, the diagnostic/detection agent or the therapeutic agentis selected from the group comprising isotopes, drugs, toxins,cytokines, oligonucleotides, hormones, hormone antagonists, enzymes,enzyme inhibitors, growth factors, radionuclides, and metals.

In one embodiment, an oligonucleotide, such as an antisense moleculeinhibiting bcl-2 expression is described in U.S. Pat. No. 5,734,033(Reed), which is incorporated by reference in its entirety, may beconjugated to, or form the therapeutic agent portion of animmunoconjugate or antibody fusion protein of the present invention.Alternatively, the oligonucleotide may be administered concurrently orsequentially with a naked or conjugated PAM4 antibody or antibodyfragment of the present invention. In a preferred embodiment, theoligonucleotides is an antisense oligonucleotide that preferably isdirected against an oncogene or oncogene product of a B-cell malignancy,such as bcl-2.

Described in the present invention is a method for diagnosing ortreating cancer, comprising: (a) administering to a subject in needthereof the antibody or fragment thereof of any one of the multivalent,multispecific antibodies or fragments thereof of the present inventionthat have an affinity toward a PAM4 antigen and comprise one or morehapten binding sites; (b) waiting a sufficient amount of time for anamount of the non-bound antibody to clear the subject's blood stream;and (c) administering to said subject a carrier molecule comprising adiagnostic/detection agent, a therapeutic agent, or a combinationthereof, that binds to a binding site of the antibody. In a preferredembodiment cancer is pancreatic cancer. Also preferred, the method canbe used for intraoperative identification of diseased tissues,endoscopic identification of diseased tissues, or intravascularidentification of diseased tissues.

Another embodiment of the present invention is a method of treating amalignancy in a subject comprising: (a) administering to said subject atherapeutically effective amount of an antibody or fragment thereofcomprising a PAM4 MAb or fragment thereof or an antibody fusion proteinor fragment thereof of any one of the antibodies, fusion proteins orfragments thereof of the present invention, wherein said PAM4 MAb orfragment thereof or antibody fusion protein or fragment thereof isconjugated to at least one therapeutic agent, and (b) formulating saidPAM4 MAb or fragment thereof or antibody fusion protein or fragmentthereof in a pharmaceutically suitable excipient. Preferably, the methodfurther comprises a second MAb or fragment thereof not in any one of theantibodies, fusion proteins or fragments thereof of the presentinvention. Still preferred, the second MAb or fragment thereof is anaked MAb or fragment thereof. Also preferred, the second MAb orfragment thereof is selected from the group consisting of CA19.9,DUPAN2, SPAN1, Nd2, B72.3, CC49, CEA, aLe^(a), antibodies defined by theLewis antigen Le(y), and antibodies against CSAp, MUC1, MUC-2, MUC-3,MUC-4, TAG-72, EGFR, CD40, angiogenesis factors (e.g., VEGF),insulin-like growth factor (IGF), tenascin, platelet derived growthfactor, IL-6, products of oncogenes and HER2/neu.

Contemplated herein is a method of diagnosing a malignancy in a subjectcomprising (a) administering to said subject a diagnostically effectiveamount of a diagnostic conjugate comprising a PAM4 MAb or fragmentthereof or PAM4 antibody fusion protein or fragment thereof of any oneof the antibodies, fusion proteins or fragments thereof of the presentinvention, wherein said PAM4 MAb or fragment thereof or PAM4 antibodyfusion protein or fragment thereof is conjugated to at least onediagnostic/detection agent, and (b) optionally formulating said PAM4 MAbor fragment thereof or antibody fusion protein or fragment thereof in apharmaceutically suitable excipient.

Another embodiment of the present invention is a method of treating acancer cell in a subject comprising (i) administering to said subject atherapeutically effective amount of a composition comprising a nakedPAM4 MAb or fragment thereof or a naked antibody fusion protein orfragment thereof of any one of the naked antibodies, fusion proteins, orfragments thereof of the present invention (ii) formulating said nakedPAM4 MAb or fragment thereof or antibody fusion protein or fragmentthereof in a pharmaceutically suitable excipient. Preferably, the methodfurther comprises a second naked antibody or fragment thereof not anyone of the naked antibodies, fusion proteins or fragments thereof of thepresent invention. For example, the second antibody or fragment thereofmay be selected from the group consisting of CA19.9, DUPAN2, SPAN1, Nd2,B72.3, CC49, CEA, aLe^(a), antibodies defined by the Lewis antigenLe(y), and antibodies against CSAp, MUC1, MUC-2, MUC-3, MUC-4, TAG-72,EGFR, CD40, CD52, HLA-DR, angiogenesis factors (e.g., VEGF),insulin-like growth factor (IGF), tenascin, platelet derived growthfactor, IL-6, products of oncogenes and HER2/neu.

The present invention also describes a method of diagnosing a malignancyin a subject comprising (i) performing an in vitro diagnosis assay on aspecimen from said subject with a composition comprising a naked PAM4MAb or fragment thereof or a naked antibody fusion protein or fragmentthereof of any one of the naked antibodies, fusion proteins, orfragments thereof of the present invention. Preferably, the malignancyis a cancer. Still preferred, the cancer is pancreatic cancer.

Another embodiment of the present invention is a method ofintraoperatively identifying diseased tissues expressing PAM4 antigen,in a subject, comprising: (A) administering an effective amount of abispecific antibody or antibody fragment comprising at least one armthat specifically binds a targeted tissue expressing PAM4-antigen and atleast one other arm that specifically binds a targetable conjugate,wherein said one arm that specifically binds a targeted tissue is ahPAM4 antibody or fragment thereof; and (B) administering a targetableconjugate selected from the group consisting of:

-   -   (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂;    -   (ii) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂;    -   (iii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;

Also described herein is a method for the endoscopic identification ofdiseased tissues expressing PAM4 antigen, in a subject, comprising: (A)administering an effective amount of a bispecific antibody or antibodyfragment comprising at least one arm that specifically binds a targetedtissue expressing PAM4-antigen and at least one other arm thatspecifically binds a targetable conjugate wherein said one arm thatspecifically binds a targeted tissue is a hPAM4 antibody or fragmentthereof, and (B) administering a targetable conjugate selected from thegroup consisting of:

-   -   (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂;    -   (ii) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂;    -   (iii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;

Contemplated herein is a method for the intravascular identification ofdiseased tissues expressing PAM4 antigen, in a subject, comprising: (A)administering an effective amount of a bispecific antibody or antibodyfragment comprising at least one arm that specifically binds a targetedtissue expressing PAM4-antigen and at least one other arm thatspecifically binds a targetable conjugate wherein said one arm thatspecifically binds a targeted tissue is a hPAM4 antibody or fragmentthereof, and (B) administering a targetable conjugate selected from thegroup consisting of:

-   -   (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂;    -   (ii) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂;    -   (iii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;

Another embodiment is a method of detection of lesions during anendoscopic, intravascular catheter, or surgical procedure, wherein themethod comprises: (a) injecting a subject who is to undergo such aprocedure with a bispecific antibody F(ab)₂ or F(ab′)₂ fragment thereof,diabody, triabody, or tetrabody., wherein said bispecific antibody orfragment thereof, diabody, triabody or tetrabody has a first antibodybinding site which specifically binds to a PAM4 antigen, and has asecond antibody binding site which specifically binds to a hapten, andpermitting the antibody fragment to accrete at target sites; (b)optionally clearing non-targeted antibody fragments using agalactosylated anti-idiotype clearing agent if the bispecific fragmentis not largely cleared from circulation within about 24 hours ofinjection, and injecting a bivalent labeled hapten, which quicklylocalizes at the target site and clears through the kidneys; (c)detecting the presence of the hapten by close-range detection ofelevated levels of accreted label at the target sites with detectionmeans, within 48 hours of the first injection, and conducting saidprocedure, wherein said detection is performed without the use of acontrast agent or subtraction agent.

A method for close-range lesion detection, during an operative,intravascular, or endoscopic procedure, wherein the method comprises:(a) injecting a subject to such a procedure parenterally with aneffective amount of a hPAM4 immunoconjugate or fragment thereof, (b)conducting the procedure within 48 hours of the injection; (c) scanningthe accessed interior of the subject at close range with a detectionmeans for detecting the presence of said labeled antibody or fragmentthereof; and (d) locating the sites of accretion of said labeledantibody or fragment thereof by detecting elevated levels of saidlabeled antibody or fragment thereof at such sites with the detectionmeans, is also considered in the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the cloned V genes and the deduced amino acid sequences ofthe murine PAM4. FIG. 1A shows the DNA and amino acid sequences of thePAM4 Vk. FIG. 1B shows the DNA and amino acid sequences of the PAM4VH.Amino acid sequences encoded by the corresponding DNA sequences aregiven as one-letter codes below the nucleotide sequence. Numbering ofthe nucleotide sequence is on the right side. The amino acid residues inthe CDR regions are shown in bold and underlined. Kabat's Ig moleculenumbering is used for amino acid residues as shown by the numberingabove the amino acid residues. The amino acid residues numbered by aletter are the insertion residues defined by Kabat numbering scheme. Theinsertion residues have the same preceding digits as that of theprevious residue. For example, residues 82, 82A, 82B, and 82C in FIG. 1Bare indicated as 82, A, B, and C, respectively.

FIG. 2 shows the amino acid sequences of the chimeric PAM4 (cPAM4) heavyand light chain variable regions expressed in Sp2/0 cells. FIG. 2A showsthe amino acid sequence of the cPAM4Vk. FIG. 2B shows the amino acidsequence of the cPAM4VH. The sequences are given as one letter codes.The amino acid residues in the CDR regions are shown in bold andunderlined. The numbering of amno acids is same as that in FIG. 1.

FIG. 3 shows the alignment of the amino acid sequences of heavy andlight chain variable regions of a human antibody, PAM4 and hPAM4. FIG.3A shows the Vκ amino acid sequence alignment of the human antibodyWalker with PAM4 and hPAM4, and FIG. 3B shows the VH amino acid sequencealignment of the human antibody Wil2 (FR1-3) and NEWM (FR4) with PAM4and hPAM4. Dots indicate the residues of PAM4 are identical to thecorresponding residues of the human antibodies. Boxed regions representthe CDR regions. Both N- and C-terminal residues (underlined) of hPAM4are fixed by the staging vectors used. Kabat's Ig molecule number schemeis used to number the residues as in FIG. 1.

FIG. 4 shows the DNA and amino acid sequences of the humanized PAM4(hPAM4) heavy and light chain variable regions expressed in Sp2/0 cells.FIG. 4A shows the DNA and amino acid sequences of the hPAM4Vκ and FIG.4B shows the DNA and amino acid sequences of the hPAM4VH. Numbering ofthe nucleotide sequence is on the right side. Amino acid sequencesencoded by the corresponding DNA sequences are given as one-lettercodes. The amino acid residues in the CDR regions are shown in bold andunderlined. Kabat's Ig molecule numbering scheme is used for amino acidresidues as in FIG. 1A and FIG. 1B.

FIG. 5 shows the binding activity of humanized PAM4 antibody, hPAM4, ascompared to the chimeric PAM4, cPAM4. hPAM4 is shown by diamonds andcPAM4 is shown by closed circles. Results indicate comparable bindingactivity of the hPAM4 antibody and cPAM4 when competing with ¹²⁵1-cPAM4binding to CaPan1 Ag. A chimeric antibody is a recombinant protein thatcontains the variable domains including the complementarity determiningregions (CDRs) of an antibody derived from one species while theconstant domains of the antibody molecule is derived from those of ahuman antibody.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Overview

Unless otherwise specified, “a” or “an” means “one or more.” Asdescribed herein, the term “PAM4 antibody” includes murine, human, andhumanized PAM4 antibodies.

The present invention relates to a monoclonal antibody, PAM4, that isuseful for the diagnosis, detection, staging, and therapy of pancreaticcancer. Preferably, the PAM4 antibodies and fragments thereof of thepresent invention are humanized or fully human. The murine PAM4 (mPAM4)antibody is a MUC1 antibody developed by employing a pancreatic cancermucin derived from the xenografted RIP-1 human pancreatic carcinoma asimmunogen. Gold et al., Int. J. Cancer, 57:204-210 (1994). The mPAM4antibody recognizes a unique and novel epitope on the target pancreaticcancer antigen. Immunohistochemical staining studies, such as thosedescribed in Example 1, have shown that the PAM4 MAb binds the domainlocated between the amino terminus and start of the repeat domain of aMUC1 antigen expressed by breast, pancreas and other cancer cells, withlimited binding to normal human tissue. The PAM4 antibodies of thepresent invention are relatively specific to pancreatic cancer andtherefore preferentially bind pancreatic cancer cells. In a preferredembodiment, the PAM4 antibodies and fragments thereof are humanized. ThePAM4 antibody is reactive with a target epitope, which can be rapidlyinternalized. This epitope is expressed primarily by antigens associatedwith pancreatic cancer and not with focal pancreatitis. Localization andtherapy studies using a radiolabeled PAM4 MAb in animal models havedemonstrated tumor targeting and therapeutic efficacy.

The PAM4 antibodies of the present invention bind the PAM4 antigen,which is the domain located between the amino terminus and start of therepeat domain of MUC 1, an antigen produced by many organs and tumortypes. A preferred PAM4 antibody of the present invention preferentiallybinds pancreatic cancer cells. Studies with a PAM4 MAb, such as the PAM4monoclonal antibody in Example 2, indicate that the antibody exhibitsseveral important properties, which make it a candidate for clinicaldiagnostic and therapeutic applications. Since the PAM4 antigen providesa useful target for diagnosis and therapy, it is desirable to obtain aMAb that recognizes an epitope of a pancreatic cancer antigen that isdistinct from the epitopes recognized by the non-PAM4 antibodies(CA19.9, DUPAN2, SPAN1, Nd2, B72.3, aLe^(a), and the Lewis antigens)described in earlier studies.

Antibodies suitable for use in combination or conjunction with the PAM4antibodies of the present invention include, for example, the antibodiesCA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, CEA, aLe^(a), and antibodiesdefined by the Lewis antigen Le(y), or antibodies againstcarcinoembryonic antigen (CEA), colon-specific antigen-p (CSAp), MUC1,MUC2, MUC3, MUC4, HER2/neu, EGFR, angiogenesis factors (e.g., VEGF),insulin-like growth factor (IGF), tenascin, platelet derived growthfactor, and IL-6, as well as products of oncogenes, and antibodiesagainst tumor necrosis substances, such as described in patents byEpstein et al. (U.S. Pat. Nos. 6,071,491, 6.017,514, 5,019,368 and5,882,626). Such antibodies would be useful for complementing currentPAM4 antibody immunodetection and immunotherapy methods. In therapyapplications, antibodies that are agonistic or antagonistic toimmunomodulators involved in effector cell function against tumor cellscould also be useful in combination with PAM4 antibodies alone or incombination with other tumor-associated antibodies, one example beingantibodies against CD40. Todryk et al., J. Immunol Methods, 248:139-147(2001); Turner et al., J. Immunol, 166:89-94 (2001). Also of use areantibodies against markers or products of oncogenes, or antibodiesagainst angiogenesis factors, such as VEGF. VEGF antibodies aredescribed in Thorpe et al., U.S. Pat. Nos. 6,342,221, 5,965,132 and6,004,554, and are incorporated by reference in their entirety.

Moreover, the availability of another PAM4-like antibody is essentialfor the development of a double-determinant enzyme-linked immunosorbentassay (ELISA), which is useful for detecting a PAM4 antigen in clinicalsamples. ELISA experiments are described in Example 4 and 7.

The present invention describes humanized and fully human antibodies andfragments thereof that bind the domain located between the aminoterminus and start of the repeat domain of MUC1 and can be used fordiagnostic and therapeutic methods. In a preferred embodiment, the PAM4antibody is humanized. Also preferred, the PAM4 antibodies of thepresent invention preferentially bind pancreatic cancer cells. Becausenon-human monoclonal antibodies can be recognized by the human host as aforeign protein, and repeated injections can lead to harmfulhypersensitivity reactions, humanization of a murine PAM4 sequences canreduce the adverse immune response that patients may experience. Formurine-based monoclonal antibodies, this is often referred to as a HumanAnti-Mouse Antibody (HAMA) response. Preferably some human residues inthe framework regions of the humanized PAM4 antibody or fragmentsthereof are replaced by their murine counterparts. It is also preferredthat a combination of framework sequences from two different humanantibodies are used for V_(H). The constant domains of the antibodymolecule are derived from those of a human antibody.

Another preferred embodiment of the present invention is a human PAM4antibody. A human antibody is an antibody obtained, for example, fromtransgenic mice that have been “engineered” to produce specific humanantibodies in response to antigenic challenge. In this technique,elements of the human heavy and light chain locus are introduced intostrains of mice derived from embryonic stem cell lines that containtargeted disruptions of the endogenous heavy chain and light chain loci.The transgenic mice can synthesize human antibodies specific for humanantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al., Nature Genet. 7:13 (1994), Lonberg etal., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994).A fully human antibody also can be constructed by genetic or chromosomaltransfection methods, as well as phage display technology, all of whichare known in the art. See for example, McCafferty et al., Nature348:552-553 (1990) for the production of human antibodies and fragmentsthereof in vitro, from immunoglobulin variable domain gene repertoiresfrom unimmunized donors. In this technique, antibody variable domaingenes are cloned in-frame into either a major or minor coat protein geneof a filamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, fortheir review, see e.g. Johnson and Chiswell, Current Opinion inStructural Biology 3:5564-571 (1993).

The antibodies and fragments thereof of the present invention arepreferably raised against a crude mucin preparation from a tumor of thehuman pancreas. In a related vein, the PAM4 antibody can be obtainedusing a substantially pure preparation of the PAM4 antigen. Asubstantially pure protein is a protein that is essentially free fromcontaminating cellular components, which are associated with the proteinin nature.

Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of thepresent invention.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, sFv and the like. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by thefull-length antibody. For example, an anti-CD20 monoclonal antibodyfragment binds with an epitope of CD20. The term “antibody fragment”also includes any synthetic or genetically engineered protein that actslike an antibody by binding to a specific antigen to form a complex. Forexample, antibody fragments include isolated fragments consisting of thevariable regions, such as the “Fv” fragments consisting of the variableregions of the heavy and light chains, recombinant single chainpolypeptide molecules in which light and heavy variable regions areconnected by a peptide linker (“scFv proteins”), and minimal recognitionunits consisting of the amino acid residues that mimic the hypervariableregion.

A naked antibody is generally an antibody that is not conjugated to atherapeutic or diagnostic/detection agent. However, it may also be anantibody fragment that is not conjugated to a diagnostic/detection ortherapeutic agent. This is so because the Fc portion of the antibodymolecule provides effector functions, such as complement fixation andADCC, (antibody dependent cell cytotoxicity), which set mechanisms intoaction that may result in cell lysis. However, it is possible that theFc portion is not required for therapeutic function, with othermechanisms, such as apoptosis, coming into play. Naked antibodiesinclude both polyclonal and monoclonal antibodies, as well as fusionproteins and certain recombinant antibodies, such as chimeric, humanizedor human antibodies.

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule are derived from those ofa human antibody. For veterinary applications, the constant domains ofthe chimeric antibody may be derived from that of other species, such asa cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, are transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains. The constant domains of the antibodymolecule are derived from those of a human antibody.

A human antibody is an antibody obtained from transgenic mice that havebeen “engineered” to produce specific human antibodies in response toantigenic challenge. In this technique, elements of the human heavy andlight chain loci are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see e.g. Johnsonand Chiswell, Current Opiniion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incoporated intheir entirety by reference.

A therapeutic agent is a molecule or atom which is administeredseparately, concurrently or sequentially with an antibody moiety orconjugated to an antibody moiety, i.e., antibody or antibody fragment,or a subfragment, and is useful in the treatment of a disease. Examplesof therapeutic agents include antibodies, antibody fragments, drugs,toxins, nucleases, hormones, immunomodulators, chelators, boroncompounds, photoactive agents or dyes and radioisotopes.

A diagnostic/detection agent is a molecule or atom which is administeredconjugated to an antibody moiety, i.e., antibody or antibody fragment,or subfragment, and is useful in diagnosing a disease by locating thecells containing the antigen. Useful diagnostic/detection agentsinclude, but are not limited to, radioisotopes, dyes (such as with thebiotin-streptavidin complex), contrast agents, fluorescent compounds ormolecules and enhancing agents (e.g. paramagnetic ions) for magneticresonance imaging (MRI). U.S. Pat. No. 6,331,175 describes MRI techniqueand the preparation of antibodies conjugated to a MRI enhancing agentand is incoporated in its entirety by reference. Preferably, thediagnostic/detection agents are selected from the group consisting ofradioisotopes, enhancing agents for use in magnetic resonance imaging,and fluorescent compounds. In order to load an antibody component withradioactive metals or paramagnetic ions, it may be necessary to react itwith a reagent having a long tail to which are attached a multiplicityof chelating groups for binding the ions. Such a tail can be a polymersuch as a polylysine, polysaccharide, or other derivatized orderivatizable chain having pendant groups to which can be boundchelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crownethers, bis-thiosemicarbazones, polyoximes, and like groups known to beuseful for this purpose. Chelates are coupled to the antibodies usingstandard chemistries. The chelate is normally linked to the antibody bya group, which enables formation of a bond to the molecule with minimalloss of immunoreactivity and minimal aggregation and/or internalcross-linking. Other, more unusual, methods and reagents for conjugatingchelates to antibodies are disclosed in U.S. Pat. No. 4,824,659 toHawthorne, entitled “Antibody Conjugates”, issued Apr. 25, 1989, thedisclosure of which is incorporated herein in its entirety by reference.Particularly useful metal-chelate combinations include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, used with diagnostic isotopes inthe general energy range of 60 to 4,000 keV, such as ¹²⁵I, ¹³¹I, ¹²³I,¹²⁴I, ⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸ Ga, ^(99m)Tc, ^(94m)Tc, ¹¹C, ¹³N,¹⁵O, ⁷⁶Br, for radio-imaging. The same chelates, when complexed withnon-radioactive metals, such as manganese, iron and gadolinium areuseful for MRI, when used along with the antibodies of the invention.Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with avariety of metals and radiometals, most particularly with radionuclidesof gallium, yttrium and copper, respectively. Such metal-chelatecomplexes can be made very stable by tailoring the ring size to themetal of interest. Other ring-type chelates such as macrocyclicpolyethers, which are of interest for stably binding nuclides, such as²²³Ra for RAIT are encompassed by the invention.

An immunoconjugate is an antibody, fusion protein, or fragment thereofconjugated to at least one therapeutic and/or diagnostic/detectionagent. The diagnostic/detection agent can comprise a radionuclide ornon-radionuclide, a contrast agent (such as for magnetic resonanceimaging, computed tomography or ultrasound), and the radionuclide can bea gamma-, beta-, alpha-, Auger electron-, or positron-emitting isotope.

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells, as well as transgenicanimals, that have been genetically engineered to contain the clonedgene(s) in the chromosome or genome of the host cell or cells of thehost cells. Suitable mammalian host cells include mycloma cells, such asSP2/0 cells, and NS0 cells, as well as Chinese Hamster Ovary (CHO)cells, hybridoma cell lines and other mammalian host cell useful forexpressing antibodies. Also particularly useful to express mAbs andother fusion proteins, is a human cell line, PER.C6 disclosed in WO0063403 A2, which produces 2 to 200-fold more recombinant protein ascompared to conventional mammalian cell lines, such as CHO, COS, Vero,Hela, BHK and SP2— cell lines. Special transgenic animals with amodified immune system are particularly useful for making fully humanantibodies.

As used herein, the term antibody fusion protein is a recombinantlyproduced antigen-binding molecule in which two or more of the same ordifferent natural antibody, single-chain antibody or antibody fragmentsegments with the same or different specificities are linked. Valency ofthe fusion protein indicates the total number of binding arms or sitesthe fusion protein has to an antigen or epitope; i.e., monovalent,bivalent, trivalent or mutlivalent. The multivalency of the antibodyfusion protein means that it can take advantage of multiple interactionsin binding to an antigen, thus increasing the avidity of binding to theantigen. Specificity indicates how many antigens or epitopes an antibodyfusion protein is able to bind; i.e., monospecific, bispecific,trispecific, multispecific. Using these definitions, a natural antibody,e.g., an IgG, is bivalent because it has two binding arms but ismonospecific because it binds to one antigen. Monospecific, multivalentfusion proteins have more than one binding site for an epitope but onlybind with the same or different epitopes on the same antigen, forexample a diabody with two binding sites reactive with the same antigen.The fusion protein may comprise a multivalent or multispecificcombination of different antibody components or multiple copies of thesame antibody component. The fusion protein may additionally comprise atherapeutic agent. Examples of therapeutic agents suitable for suchfusion proteins include immunomodulators (“antibody-immunomodulatorfusion protein”) and toxins (“antibody-toxin fusion protein”). Onepreferred toxin comprises a ribonuclease (RNase), preferably arecombinant RNase.

A multispecific antibody is an antibody that can bind simultaneously toat least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. One specificity would be for aB-cell, T-cell, myeloid-, plasma-, and mast-cell antigen or epitope.Another specificity could be to a different antigen on the same celltype, such as CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, and CD22on B-cells. Multispecific, multivalent antibodies are constructs thathave more than one binding site, and the binding sites are of differentspecificity. For example, a diabody, where one binding site reacts withone antigen and the other with the another antigen.

A bispecific antibody is an antibody that can bind simultaneously to twotargets which are of different structure. Bispecific antibodies (bsAb)and bispecific antibody fragments (bsFab) have at least one arm thatspecifically binds to, for example, a B-cell, T-cell, myeloid-, plasma-,and mast-cell antigen or epitope and at least one other arm thatspecifically binds to a targetable conjugate that bears a therapeutic ordiagnostic/detection agent. A variety of bispecific fusion proteins canbe produced using molecular engineering. In one form, the bispecificfusion protein is monovalent, consisting of, for example, a scFv with asingle binding site for one antigen and a Fab fragment with a singlebinding site for a second antigen. In another form, the bispecificfusion protein is divalent, consisting of, for example, an IgG with abinding site for one antigen and two scFv with two binding sites for asecond antigen.

Preparation of Humanized and Human PAM4 Antibodies

Monoclonal antibodies for specific antigens may be obtained by methodsknown to those skilled in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991) (hereinafter “Coligan”). Briefly, PAM4 MAbs can be obtained byinjecting mice with a composition comprising the PAM4 antigen, verifyingthe presence of antibody production by removing a serum sample, removingthe spleen to obtain B-lymphocytes, fusing the B-lymphocytes withmyeloma cells to produce hybridomas, cloning the hybridomas, selectingpositive clones which produce antibodies to PAM4 antigen, culturing theclones that produce antibodies to PAM4 antigen, and isolating PAM4antibodies from the hybridoma cultures. The PAM4 antibodies of thepresent invention bind the PAM4 antigen, a domain located between theamino terminus and the start of the repeat domain of MUC 1. The PAM4antibodies of the present invention preferentially bind pancreaticcancer cells.

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization of murine antibodies and antibody fragments is well knownto those skilled in the art. For example, humanized monoclonalantibodies are produced by transferring murine complementary determiningregions from heavy and light variable chains of the mouse immunoglobulininto a human variable domain, and then, substituting human residues inthe framework regions of the murine counterparts. The use of antibodycomponents derived from humanized monoclonal antibodies obviatespotential problems associated with the immunogenicity of murine constantregions.

A fully human antibody of the present invention, i.e., human PAM4 can beobtained from a transgenic non-human animal. See, e.g., Mendez et al.,Nature Genetics, 15: 146-156 (1997); U.S. Pat. No. 5,633,425, which areincorporated in their entirety by reference. For example, a humanantibody can be recovered from a transgenic mouse possessing humanimmunoglobulin loci. The mouse humoral immune system is humanized byinactivating the endogenous immunoglobulin genes and introducing humanimmunoglobulin loci. The human immunoglobulin loci are exceedinglycomplex and comprise a large number of discrete segments which togetheroccupy almost 0.2% of the human genome. To ensure that transgenic miceare capable of producing adequate repertoires of antibodies, largeportions of human heavy- and light-chain loci must be introduced intothe mouse genome. This is accomplished in a stepwise process beginningwith the formation of yeast artificial chromosomes (YACs) containingeither human heavy- or light-chain immunoglobulin loci in germlineconfiguration. Since each insert is approximately 1 Mb in size, YACconstruction requires homologous recombination of overlapping fragmentsof the immunoglobulin loci. The two YACs, one containing the heavy-chainloci and one containing the light-chain loci, are introduced separatelyinto mice via fusion of YAC-containing yeast spheroblasts with mouseembryonic stem cells. Embryonic stem cell clones are then microinjectedinto mouse blastocysts. Resulting chimeric males are screened for theirability to transmit the YAC through their germline and are bred withmice deficient in murine antibody production. Breeding the twotransgenic strains, one containing the human heavy-chain loci and theother containing the human light-chain loci, creates progeny whichproduce human antibodies in response to immunization.

General techniques for cloning murine immunoglobulin variable domainsare described, for example, by the publication of Orlandi et al., Proc.Nat'l Acad. Sci. USA 86: 3833 (1989), which is incorporated by referencein its entirety. Techniques for producing humanized MAbs are described,for example, by Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285(1992), Singer et al., J. Immun. 150: 2844 (1992), Mountain et al.Biotechnol. Genet. Eng. Rev. 10: 1 (1992), and Coligan at pages10.19.1-10.19.11, each of which is hereby incorporated by reference.

In general, the V_(κ) (variable light chain) and V_(H) (variable heavychain) sequences for PAM4 antibodies can be obtained by a variety ofmolecular cloning procedures, such as RT-PCR, 5′-RACE, and cDNA libraryscreening. Specifically, the V_(H) and V_(κ) genes of the MAb PAM4 werecloned by PCR amplification from the hybridoma cells by RT-PCR and 5′RACE, respectively, and their sequences determined by DNA sequencing. Toconfirm their authenticity, the cloned V_(L) and V_(H) genes can beexpressed in cell culture as an Ab as described by Orlandi et al.,(Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)) which is incorporated byreference. Based on the V gene sequences, a humanized PAM4 antibody canthen be designed and constructed as described by Leung et al. (Mol.Immunol., 32: 1413 (1995)), which is incorporated by reference. cDNA canbe prepared from any known hybridoma line or transfected cell lineproducing a murine or humanized PAM4 antibody by general molecularcloning techniques (Sambrook et al., Molecular Cloning, A laboratorymanual, 2^(nd) Ed (1989)). Example 7 describes the humanization processutilized for hPAM4 MAb.

Antibodies can generally be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof humanized antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2μ membrane. The filtered medium is passed througha protein A column (1×3 cm) at a flow rate of 1 ml/min. The resin isthen washed with about 10 column volumes of PBS and protein A-boundantibody is eluted from the column with 0.1 M glycine buffer (pH 3.5)containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3 M Tris (pH 8.6), and protein concentrationsdetermined from the absorbance at 280/260 nm. Peak fractions are pooled,dialyzed against PBS, and the antibody concentrated, for example, withthe Centricon 30 (Amicon, Beverly, Mass.). The antibody concentration isdetermined by ELISA, as before, and its concentration adjusted to about1 mg/ml using PBS. Sodium azide, 0.01% (w/v), is conveniently added tothe sample as preservative.

The nucleotide sequences of the primers used to prepare the PAM4antibodies are discussed in Example 7, below. In a preferred embodiment,a humanized PAM4 antibody or antibody fragment comprises thecomplementarity-determining regions (CDRs) of a murine PAM4 MAb and theframework (FR) regions of the light and heavy chain variable regions ofa human antibody and the light and heavy chain constant regions of ahuman antibody, wherein the CDRs of the light chain variable region ofthe humanized PAM4 comprises CDR1 comprising an amino acid sequence ofSASSSVSSSYLY; CDR2 comprising an amino acid sequence of STSNLAS; andCDR3 comprising an amino acid sequence of HQWNRYPYT; and the CDRs of theheavy chain variable region of the humanized PAM4 MAb comprises CDR1comprising an amino acid sequence of SYVLH; CDR2 comprising an aminoacid sequence of YINPYNDGTQYNEKFKG and CDR3 comprising an amino acidsequence of GFGGSYGFAY. Also preferred, the FRs of the light and heavychain variable regions of the humanized antibody comprise at least oneamino acid substituted from said corresponding FRs of the murine PAM4MAb.

PAM4 MAbs can be isolated and purified from hybridoma cultures by avariety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

PAM4 MAbs can be characterized by a variety of techniques that arewell-known to those of skill in the art. For example, the ability of aPAM4 MAb to bind to the PAM4 antigen can be verified using an indirectenzyme immunoassay, flow cytometry analysis, or Western analysis.

Production of PAM4 Antibody Fragments

The present invention contemplates the use PAM4 antibody fragments.Antibody fragments which recognize specific epitopes can be generated byknown techniques. The antibody fragments are antigen binding portions ofan antibody, such as F(ab′)₂, Fab′, Fab, Fv, sFv and the like. F(ab′)₂fragments, for example, can be produced by pepsin digestion of theantibody molecule and Fab′ fragments can be generated by reducingdisulfide bridges of the F(ab)′2 fragments. These methods are described,for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 andreferences contained therein, which patents are incorporated herein intheir entireties by reference. Also, see Nisonoff et al., Arch Biochem.Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959), Edelman etal., in METHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967),and Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4. Alternatively, Fab′expression libraries can be constructed (Huse et al., 1989, Science,246:1274-1281) to allow rapid and easy identification of monoclonal Fab′fragments with the desired specificity. The present inventionencompasses antibodies and antibody fragments.

A single chain Fv molecule (scFv) comprises a V_(L) domain and a V_(H)domain. The V_(L) and V_(H) domains associate to form a target bindingsite. These two domains are further covalently linked by a peptidelinker (L). A scFv molecule is denoted as either V_(L)-L-V_(H) if theV_(L) domain is the N-terminal part of the scFv molecule, or asV_(H)-L-V_(L) if the V_(H) domain is the N-terminal part of the scFvmolecule. Methods for making scFv molecules and designing suitablepeptide linkers are described in U.S. Pat. No. 4,704,692, U.S. Pat. No.4,946,778, R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80(1995) and R. E. Bird and B. W. Walker, “Single Chain Antibody VariableRegions,” TIBTECH, Vol 9: 132-137 (1991). These references areincorporated herein by reference.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull-length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full-length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide an approximate 100 Kdfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent, and optionally a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce anapproximate 50 Kd Fab′ monovalent fragment. Alternatively, an enzymaticcleavage using papain produces two monovalent Fab fragments and an Fefragment directly. These methods are described, for example, byGoldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 and referencescontained therein, which patents are incorporated herein in theirentireties by reference. Also, see Nisonoff et al., Arch Biochem.Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959), Edelman etal., in METHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967),and Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). A CDR is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to ashypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction (PCR) to synthesize the variable region fromRNA of antibody-producing cells. See, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2: 106 (1991);Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION,Ritter et al. (eds.), pages 166-179 (Cambridge University Press 1995);and Ward et al., “Genetic Manipulation and Expression of Antibodies,” inMONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al.,(eds.), pages 137-185 (Wiley-Liss, Inc. 1995).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Production of Humanized and Human PAM4 Antibody Fusion Proteins

The antibody fusion proteins of the present invention can be prepared bya variety of conventional procedures, ranging from glutaraldehydelinkage to more specific linkages between functional groups. Theantibodies and/or antibody fragments that comprise the fusion proteinsdescribed herein are preferably covalently bound to one another,directly or through a linker moiety, through one or more functionalgroups on the antibody or fragment, e.g., amine, carboxyl, phenyl,thiol, or hydroxyl groups. Various conventional linkers in addition toglutaraldehyde can be used, e.g., diisocyanates, diiosothiocyanates,bis(hydroxysuccinimide)esters, carbodiimides,maleimidehydroxysuccinimide esters, and the like.

A simple method for producing humanized and human PAM4 fusion proteinsis to mix the antibodies or fragments in the presence of glutaraldehyde.The initial Schiff base linkages can be stabilized, e.g., by borohydridereduction to secondary amines. A diiosothiocyanate or carbodiimide canbe used in place of glutaraldehyde as a non-site-specific linker. In oneembodiment of the present invention, an antibody fusion proteincomprises one humanized or human PAM4 MAb, or fragment thereof, whereinthe MAb binds to the domain located between the amino terminus and thestart of the repeat domain of the MUC1 antigen. This fusion protein andfragments thereof preferentially bind pancreatic cancer cells. Thismonovalent, monospecific MAb is useful for direct targeting of anantigen, where the MAb is attached to a therapeutic agent, adiagnostic/detection agent, or a combination thereof, and the protein isadministered directly to a patient in need thereof.

The PAM4 antibody fusion proteins and fragments thereof of the presentinvention may instead comprise at least two humanized or human PAM4MAbs, or fragments thereof, wherein at least two of the MAbs orfragments thereof bind to distinct epitopes of the PAM4 antigen. Forexample, the MAbs can produce antigen specific diabodies, triabodies andtetrabodies, which are multivalent but monospecific to the PAM4 antigen.The non-covalent association of two or more scFv molecules can formfunctional diabodies, triabodies and tetrabodies. Monospecific diabodiesare homodimers of the same scFv, where each scFv comprises the V_(H)domain from the selected antibody connected by a short linker to theV_(L) domain of the same antibody. A diabody is a bivalent dimer formedby the non-covalent association of two scFvs, yielding two Fv bindingsites. A triabody results from the formation of a trivalent trimer ofthree scFvs, yielding three binding sites, and a tetrabody is atetravalent tetramer of four scFvs, resulting in four binding sites.Several monospecific diabodies have been made using an expression vectorthat contains a recombinant gene construct comprisingV_(H1)-linker-V_(L1). See Holliger et al., Proc. Natl. Acad. Sci. USA90: 6444-6448 (1993); Atwell et al., Molecular Immunology 33: 1301-1302(1996); Holliger et al., Nature Biotechnology 15: 632-631(1997);Helfrich et al., Int. J. Cancer 76: 232-239 (1998); Kipriyanov et al.,Int. J. Cancer 77: 763-772 (1998); Holiger et al., Cancer Research 59:2909-2916(1999)). Methods of constructing scFvs are disclosed in U.S.Pat. No. 4,946,778 (1990) and U.S. Pat. No. 5,132,405 (1992). Methods ofproducing multivalent, monospecific antibodies based on scFv aredisclosed in U.S. Pat. No. 5,837,242 (1998), U.S. Pat. No. 5,844,094(1998) and WO-98/44001 (1998). The multivalent, monospecific antibodyfusion protein binds to two or more of the same type of epitopes thatcan be situated on the same antigen or on separate antigens. Theincreased valency allows for additional interaction, increased affinity,and longer residence times. These antibody fusion proteins can beutilized in direct targeting systems, where the antibody fusion proteinis conjugated to a therapeutic agent, a diagnostic/detection agent, or acombination thereof, and administered directly to a patient in needthereof.

A preferred embodiment of the instant invention is a multivalent,multispecific antibody or fragment thereof comprising more than oneantigen binding site having an affinity toward a PAM4 target epitope andone or more additional epitopes associated with pancreatic cancerantigens. This fusion protein is multispecific because it binds at leasttwo different epitopes, which can reside on the same or differentantigens. For example, the fusion protein may comprise more than oneantigen binding site, the first with an affinity toward one PAM4 antigenepitope and the second with an affinity toward another target antigensuch as TAG-72 or CEA. Another example is a bispecific PAM4 antibodyfusion protein which may comprise a CA19.9 MAb (or fragment thereof) anda PAM4 MAb (or fragment thereof). Such a fusion protein will have anaffinity toward CA19.9 as well as the domain located between the aminoterminus and start of the repeat domain of MUC1. Also contemplated inthe present invention is a fusion protein comprising more than oneantigen binding site having an affinity for at least two different PAM4antigen epitopes.

The antibody fusion proteins and fragments thereof of the presentinvention can be utilized in direct targeting systems, where theantibody fusion protein is conjugated to a therapeutic agent, adiagnostic/detection agent, or a combination thereof, and administereddirectly to a patient in need thereof.

Another preferred embodiment of the instant invention is a multivalent,multispecific antibodies and fragments thereof comprising more than oneantigen binding site having affinity toward a PAM4 target epitope and atleast one hapten binding site having affinity towards hapten molecules.For example, a bispecific PAM4 antibody fusion protein may comprise the679 MAb (or fragment thereof) and the PAM4 MAb (or fragment thereof).The monoclonal antibody, 679, binds with high affinity to moleculescontaining the tri-peptide moiety histamine succinyl glycyl (HSG). Sucha bispecific PAM4 antibody fusion protein can be prepared, for example,by obtaining an F(ab′)₂ fragment from 679, as described above. Theinterchain disulfide bridges of the 679 F(ab′)₂ fragment are gentlyreduced with cystine, taking care to avoid light-heavy chain linkage, toform Fab′-SH fragments. The SH group(s) is (are) activated with anexcess of bis-maleimide linker(1,1′-(methylenedi-4,1-phenylene)bis-malemide). The PAM4 MAb isconverted to Fab′-SH and then reacted with the activated MR23 Fab′-SHfragment to obtain a bispecific PAM4 antibody fusion protein. Bispecificantibody fusion proteins such as this one can be utilized in affinityenhancing systems, where the target antigen is pretargeted with thefusion protein and is subsequently targeted with a diagnostic ortherapeutic agent that binds with the antibody-antigen complex formed bypretargeting.

Bispecific antibodies can be made by a variety of conventional methods,e.g., disulfide cleavage and reformation of mixtures of whole IgG or,preferably F(ab′)₂ fragments, fusions of more than one hybridoma to formpolyomas that produce antibodies having more than one specificity, andby genetic engineering. Bispecific antibody fusion proteins have beenprepared by oxidative cleavage of Fab′ fragments resulting fromreductive cleavage of different antibodies. This is advantageouslycarried out by mixing two different F(ab′)₂ fragments produced by pepsindigestion of two different antibodies, reductive cleavage to form amixture of Fab′ fragments, followed by oxidative reformation of thedisulfide linkages to produce a mixture of F(ab′)₂ fragments includingbispecific antibody fusion proteins containing a Fab′ potion specific toeach of the original epitopes. General techniques for the preparation ofantibody fusion proteins may be found, for example, in Nisonoff et al.,Arch Biochem. Biophys. 93: 470 (1961), Hämmerling et al., J. Exp. Med.128: 1461 (1968), and U.S. Pat. No. 4,331,647. Contemplated in thepresent invention is an antibody fusion protein or fragment thereofcomprising at least one first PAM4 MAb or fragment thereof and at leastone second MAb or fragment thereof, other than the PAM4 MAbs orfragments thereof of the present invention.

More selective linkage can be achieved by using a heterobifunctionallinker such as maleimidehydroxysuccinimide ester. Reaction of the esterwith an antibody or fragment will derivatize amine groups on theantibody or fragment, and the derivative can then be reacted with, e.g.,and antibody Fab fragment having free sulfhydryl groups (or, a largerfragment or intact antibody with sulfhydryl groups appended thereto by,e.g., Traut's Reagent). Such a linker is less likely to crosslink groupsin the same antibody and improves the selectivity of the linkage.

It is advantageous to link the antibodies or fragments at sites remotefrom the antigen binding sites. This can be accomplished by, e.g.,linkage to cleaved interchain sulfydryl groups, as noted above. Anothermethod involves reacting an antibody having an oxidized carbohydrateportion with another antibody that has at lease one free amine function.This results in an initial Schiff base (mime) linkage, which ispreferably stabilized by reduction to a secondary amine, e.g., byborohydride reduction, to form the final composite. Such site-specificlinkages are disclosed, for small molecules, in U.S. Pat. No. 4,671,958,and for larger addends in U.S. Pat. No. 4,699,784—incorporated byreference.

A polyspecific PAM4 antibody fusion protein can be obtained by addingPAM4 antigen binding moieties to a bispecific humanized or human PAM4antibody fusion protein. For example, a bispecific antibody fusionprotein can be reacted with 2-iminothiolane to introduce one or moresulfhydryl groups for use in coupling the bispecific fusion protein to athird PAM4 MAb or fragment, using the bis-maleimide activation proceduredescribed above. These techniques for producing antibody fusion proteinsare well known to those of skill in the art. See, for example, U.S. Pat.No. 4,925,648, which is incorporated by reference in its entirety.

ScFvs with linkers greater than 12 amino acid residues in length (forexample, 15-or 18-residue linkers) allow interacting between the VH andVL domains on the same chain and generally form a mixture of monomers,dimers (termed diabodies) and small amounts of higher mass multimers,(Kortt et al., Eur. J. Biochem. (1994) 221: 151-157). ScFvs with linkersof 5 or less amino acid residues, however, prohibit intramolecularpairing of the V_(H) and V_(L) domains on the same chain, forcingpairing with V_(H) and V_(L) domains on a different chain. Linkersbetween 3- and 12-residues form predominantly dimers (Atwell et al.,Protein Engineering (1999) 12: 597-604). With linkers between 0 and 2residues, trimeric (termed triabodies), tetrameric (termed tetrabodies)or higher oligomeric structures of scFvs are formed; however, the exactpatterns of oligomerization appear to depend on the composition as wellas the orientation of the V-domains, in addition to the linker length.For example, scFvs of the anti-neuraminidase antibody NC10 formedpredominantly trimers (V_(H) to V_(L) orientation) or tetramers (V_(L)to V_(H) orientation) with O-residue linkers (Dolezal et al., ProteinEngineering (2000) 13: 565-574). For scFvs constructed from NC10 with 1-and 2-residue linkers, the V_(H) to V_(L) orientation formedpredominantly diabodies (Atwell et al., Protein Engineering (1999) 12:597-604); in contrast, the V_(L) to V_(H) orientation formed a mixtureof tetramers, trimers, dimers, and higher mass multimers (Dolezal etal., Protein Engineering (2000) 13: 565-574). For scFvs constructed fromthe anti-CD19 antibody HD37 in the V_(H) to V_(L) orientation, theO-residue linker formed exclusively trimers and the 1-residue linkerformed exclusively tetramers (Le Gall et al., FEBS Letters (1999) 453:164-168).

Expression Vectors and Host Cells

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements, and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.A promoter is a DNA sequence that directs the transcription of astructural gene. A structural gene is a DNA sequence that is transcribedinto messenger RNA (mRNA) which is then translated into a sequence ofamino acids characteristic of a specific polypeptide. Typically, apromoter is located in the 5′ region of a gene, proximal to thetranscriptional start site of a structural gene. If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. An enhancer is a DNA regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

An isolated DNA molecule is a fragment of DNA that is not integrated inthe genomic DNA of an organism. For example, a cloned PAM4 antigen geneis a DNA fragment that has been separated from the genomic DNA of amammalian cell. Another example of an isolated DNA molecule is achemically-synthesized DNA molecule that is not integrated in thegenomic DNA of an organism. Complementary DNA (cDNA) is asingle-stranded DNA molecule that is formed from an mRNA template by theenzyme reverse transcriptase. Typically, a short synthetic oligonucleotide complementary to a portion of the mRNA is employed as aprimer for the initiation of reverse transcription to generate the firststand DNA. Those skilled in the art also use the term “cDNA” to refer toa double-stranded DNA molecule consisting of such a single-stranded DNAmolecule and its complementary DNA strand.

A cloning vector is a DNA molecule, such as a plasmid, cosmid, orbacteriophage, that has the capability of replicating autonomously in ahost cell. Cloning vectors typically contain one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss of anessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide tetracycline resistance or ampicillin resistance. Arecombinant host may be any prokaryotic or eukaryotic cell that containseither a cloning vector or expression vector. This term also includesthose prokaryotic or eukaryotic cells that have been geneticallyengineered to contain the cloned gene(s) in the chromosome or genome ofthe host cell. The term expression refers to the biosynthesis of a geneproduct. For example, in the case of a structural gene, expressioninvolves transcription of the structural gene into mRNA and thetranslation of mRNA into one or more polypeptides.

Suitable host cells include microbial or mammalian host cells. Apreferred host is the human cell line, PER.C6, which was developed forproduction of MAbs, and other fusion proteins. Accordingly, a preferredembodiment of the present invention is a host cell comprising a DNAsequence encoding the PAM4 MAb, conjugate, fusion protein or fragmentsthereof. PER.C6 cells (WO 97/00326) were generated by transfection ofprimary human embryonic retina cells, using a plasmid that contained theAdserotype 5 (Ad5) E1A- and E1B-coding sequences (Ad5 nucleotides459-3510) under the control of the human phosphoglycerate kinase (PGK)promoter. E1A and E1B are adenovirus early gene activation protein 1Aand 1B, respectively. The methods and compositions are particularlyuseful for generating stable expression of human recombinant proteins ofinterest that are modified post-translationally, e.g. by glycosylation.Several features make PER.C6 particularly useful as a host forrecombinant protein production, such as PER.C6 is a fully characterizedhuman cell line and it was developed in compliance with good laboratorypractices. Moreover, PER.C6 can be grown as a suspension culture indefined serum-free medium devoid of any human- or animal-derivedproteins and its growth is compatible with roller bottles, shakerflasks, spinner flasks and bioreactors with doubling times of about 35hours. Finally, the presence of E1A causes an up regulation ofexpression of genes that are under the control of the CMVenhancer/promoter and the presence of E13 prevents p53-dependentapoptosis possibly enhanced through over expression of the recombinanttransgene. In one embodiment, the cell is capable of producing 2 to200-fold more recombinant protein and/or proteinaceous substance thanconventional mammalian cell lines.

Humanized and Human PAM4 Antibodies Use for Treatment and Diagnosis

Contemplated in the present invention is a method of diagnosing ortreating a malignancy in a subject comprising administering to thesubject a therapeutically effective amount of a therapeutic conjugatecomprising a PAM4 MAb or fragment thereof or an antibody fusion proteinor fragment thereof, wherein the PAM4 MAb or fragment thereof orantibody fusion protein or fragment thereof is bound to at least onediagnostic and/or therapeutic agent and then formulated in apharmaceutically suitable excipient. Also preferred is a method fordiagnosing or treating cancer, comprising: administering a multivalent,multispecific antibody or fragment thereof comprising one or moreantigen binding sites toward a PAM4 antigen and one or more haptenbinding sites to a subject in need thereof, waiting a sufficient amountof time for an amount of the non-antibody to clear the subject's bloodstream; and then administering to the subject a carrier moleculecomprising a diagnostic/detection agent, a therapeutic agent, or acombination thereof, that binds to the binding site of the multivalent,multispecific antibody or fragment thereof. In a preferred embodiment,the cancer is a pancreatic cancer. In another preferred embodiment, theantibody is a multivalent, monospecific antibody or fragment thereof.

The use of MAbs for in vitro diagnosis is well-known. See, for example,Carlsson et al., Bio/Technology 7 (6): 567 (1989). For example, MAbs canbe used to detect the presence of a tumor-associated antigen in tissuefrom biopsy samples. MAbs also can be used to measure the amount oftumor-associated antigen in clinical fluid samples using techniques suchas radioimmunoassay, enzyme-linked immunosorbent assay, and fluorescenceimmunoassay.

Conjugates of tumor-targeted MAbs and toxins can be used to selectivelykill cancer cells in vivo (Spalding, Bio/Technology 9(8): 701 (1991);Goldenberg, Scientific American Science & Medicine 1(1): 64 (1994)). Forexample, therapeutic studies in experimental animal models havedemonstrated the anti-tumor activity of antibodies carrying cytotoxicradionuclides. See Example 3 and 5 for a discussion of animal models andtherapeutic studies. (Goldenberg et al., Cancer Res. 41: 4354 (1981),Cheung et al., J. Nat'l Cancer Inst. 77: 739 (1986), and Senekowitsch etal., J. Nucl. Med. 30: 531 (1989)).

Humanized and fully human antibodies and fragments thereof are suitablefor use in therapeutic methods and diagnostic methods. Accordingly,contemplated in the present invention is a method of delivering adiagnostic or therapeutic agent, or a combination thereof, to a targetcomprising (i) providing a composition that comprises a PAM4 antibody orfragment thereof conjugated to at least one diagnostic and/ortherapeutic agent and (ii) administering to a subject in need thereofthe diagnostic or therapeutic antibody conjugate. In a preferredembodiment, the PAM4 antibodies and fragments thereof are humanized orfully human. In another embodiment, the humanized and fully human PAM4antibodies and fragments thereof of the present invention are used inmethods for treating malignancies.

Also described herein is a cancer cell targeting diagnostic ortherapeutic conjugate comprising an antibody component that comprises aPAM4 MAb or fragment thereof of any of the antibodies of the presentinvention, or an antibody fusion protein or fragment thereof, whereinthe antibody component is bound to at least one diagnostic or at leastone therapeutic agent. Preferably, the diagnostic conjugate is aphotoactive diagnostic/detection agent, an ultrasound detectable agentor an MRI contrast agent. Still preferred, the diagnostic/detectionagent is a radionuclide with an energy between 20 and 4,000 keV.

Another embodiment of the present invention is a method for diagnosingor treating a malignancy comprising administering a therapeutically ordiagnostically effective amount of at least one naked PAM4 antibody orfragment thereof and/or PAM4 fusion protein or fragment therof, andoptionally formulating the PAM4 antibody, fusion protein, or fragmentsthereof in a pharmaceutical excipient.

The compositions for treatment contain at least one humanized or fullyhuman PAM4 antibody or fragment thereof either alone and unconjugated,or conjugated or unconjugated and in combination with other antibodiesor fragments thereof, such as other humanized or chimeric antibodies,human antibodies, therapeutic agents or immunomodulators. Naked orconjugated antibodies to the same or different epitope or antigen mayalso be combined with one or more of the PAM4 antibodies or fragmentsthereof of the present invention.

Accordingly, the present invention contemplates the administration ofPAM4 antibodies and fragments thereof, including PAM4 fusion proteinsand fragments thereof, alone, as a naked antibody or antibody fragment,or administered as a multimodal therapy. Preferably, the antibody is ahumanized or fully human PAM4 antibody or fragment thereof. Multimodaltherapies of the present invention further include immunotherapy with anaked PAM4 antibody supplemented with administration of other antibodiesin the form of naked antibodies, fusion proteins, or asimmunoconjugates. For example, a humanized or fully human PAM4 antibodymay be combined with another naked humanized PAM4 or other antibody, ora humanized PAM4, or other antibody conjugated to an isotope, one ormore chemotherapeutic agents, cytokines, toxins or a combinationthereof. For example, the present invention contemplates treatment of anaked or conjugated PAM4 antibody or fragments thereof before, incombination with, or after other pancreatic tumor associated antibodiessuch as CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, la3, aLe^(a)antibodies, and other Lewis antigens (e.g., Le(y)), as well asantibodies against carcinoembryonic antigen (CEA), colon-specificantigen-p (CSAp), MUC1, MUC2, MUC3, MUC4, HER2/neu, EGFR, angiogenesisfactors (e.g., VEGF), insulin-like growth factor (IGF), tenascin,platelet derived growth factor, and IL-6, as well as products ofoncogenes and antibodies against tumor necrosis substances. These solidtumor antibodies may be naked or conjugated to, inter alia, drugs,toxins, isotopes, external radiation or immunomodulators. A fusionprotein of a humanized or fully human PAM4 antibody and a toxin or mayalso be used in this invention. Many different antibody combinations maybe constructed, either as naked antibodies or as partly naked and partlyconjugated with a therapeutic agent or immunomodulator. Alternatively,different naked antibody combinations may be employed for administrationin combination with other therapeutic agents, such as a cytotoxic drugor with radiation, given consecutively, simultaneously, or sequentially.

The monospecific antibodies described herein that are linked todiagnostic or therapeutic agents directly target PAM4 positive tumors.The monospecific molecules bind selectively to targeted antigens and asthe number of binding sites on the molecule increases, the affinity forthe target cell increases and a longer residence time is observed at thedesired location. Moreover, non-antigen bound molecules are cleared fromthe body quickly and exposure of normal tissues is minimized. A use ofmultispecific antibodies is in AES systems, where PAM4 pre-targetspositive tumors for subsequent specific delivery of diagnostic ortherapeutic agents. The agents are carried by histamine succinyl glycyl(HSG) containing peptides. The murine monoclonal antibody designated 679(an IgG1, K) binds with high affinity to molecules containing thetri-peptide moiety, HSG (Morel et al, Molecular Immunology, 27,995-1000, 1990). 679 MAb can form a bispecific antibody with hPAM4 thatbinds with HSG and the target antigen. Alternative haptens may also beutilized. These antibodies bind selectively to targeted antigensallowing for increased affinity and a longer residence time at thedesired location. Moreover, non-antigen bound diabodies are cleared fromthe body quickly and exposure of normal tissues is minimized. PAM4antibodies and fragments thereof and conjugates can be used to diagnoseand treat mammalian disorders such as cancer.

Delivering a diagnostic or a therapeutic agent to a target for diagnosisor treatment in accordance with the invention includes providing thePAM4 antibody or fragments thereof with a diagnostic or therapeuticagent and administering to a subject in need thereof with the antibody.Diagnosis further requires the step of detecting the bound proteins withknown techniques.

In the context of this application, the terms “diagnosis” or “detection”can be used interchangeably. Whereas diagnosis usually refers todefining a tissue's specific histological status, detection recognizesand locates a tissue, lesion or organism containing a particularantigen.

Administration of the antibodies and their fragments of the presentinvention with diagnostic or therapeutic agents can be effected in amammal by intravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, perfusion through a regionalcatheter, or direct intralesional injection. When administering theantibody by injection, the administration may be by continuous infusionor by single or multiple boluses.

The antibody with the diagnostic or therapeutic agent may be provided asa kit for human or mammalian therapeutic and diagnostic use in apharmaceutically acceptable injection vehicle, preferablyphosphate-buffered saline (PBS) at physiological pH and concentration.The preparation preferably will be sterile, especially if it is intendedfor use in humans. Optional components of such kits include stabilizers,buffers, labeling reagents, radioisotopes, paramagnetic compounds,second antibody for enhanced clearance, and conventional syringes,columns, vials and the like.

Naked Antibody Therapy

A therapeutically effective amount of a naked humanized and fully humanPAM4 antibody, or fragments thereof, or PAM4 fusion proteins orfragments thereof, can be formulated in a pharmaceutically acceptableexcipient. The efficacy of the naked humanized and fully human PAM4antibodies and their fragments can also be enhanced by supplementingthese naked antibodies with one or more other naked antibodies, with oneor more immunoconjugates of humanized and fully human PAM4 antibodies,conjugated with one or more therapeutic agents, including drugs, toxins,immunomodulators, hormones, oligonucleotides, hormone antagonists,enzymes, enzyme inhibitors, therapeutic radionuclides, an angiogenesisinhibitor, etc., administered concurrently or sequentially or accordingto a prescribed dosing regimen, with the PAM4 antibodies or fragmentsthereof. The naked antibodies that may supplement the naked PAM4antibodies and fragments thereof may be directed against either the sametumor type or against immunomodulator cells (e.g., CD40⁺ cells) that canbe recruited to enhance the antitumor effects of the naked antibodies ofchoice.

PAM4 Immunoconjugates

The present invention also contemplates the use of humanized and humanPAM4 antibodies and fragments thereof conjugated to at least onetherapeutic and/or diagnostic/detection agent for therapy or diagnosis.For immunotherapy, the objective is to deliver cytotoxic doses ofradioactivity, toxin, or drug to target cells, while minimizing exposureto non-target tissues. The PAM4 antibodies of the present invention canbe used to diagnose and treat pancreatic tumors.

Any of the antibodies, antibody fusion proteins, and fragments thereofof the present invention can be conjugated with one or more therapeuticor diagnostic/detection agents. Generally, one therapeutic ordiagnostic/detection agent is attached to each antibody, fusion proteinor fragment thereof but more than one therapeutic agent and/ordiagnostic/detection agent can be attached to the same antibody orantibody fragment. If the Fc region is absent (for example when theantibody used as the antibody component of the immunoconjugate is anantibody fragment), it is possible to introduce a carbohydrate moietyinto the light chain variable region of a full length antibody orantibody fragment. See, for example, Leung et al., J. Immunol. 154: 5919(1995); Hansen et al., U.S. Pat. No. 5,443,953 (1995), Leung et al.,U.S. Pat. No. 6,254,868, all of which are incorporated in their entiretyby reference. The engineered carbohydrate moiety is used to attach thetherapeutic or diagnostic/detection agent.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, all of which are incorporated in their entirety by reference.The general method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality of peptide.This reaction results in an initial Schiff base (imine) linkage, whichcan be stabilized by reduction to a secondary amine to form the finalconjugate.

The antibody fusion proteins and fragments thereof of the presentinvention comprise two or more antibodies or fragments thereof and eachof the antibodies that compose this fusion protein can contain at leastone therapeutic agent and/or diagnostic/detection agent. For example, anantibody fusion protein may comprise one antibody (two antigen bindingsites) and an antibody fragment, two antibody fragments, or twoantibodies. The antibody fusion protien may then be conjugated to atleast one diagnostic/detection and/or therapeutic agent.

Accordingly, one or more of the antibodies or fragments thereof of theantibody fusion protein can have more than one therapeutic and/ordiagnostic/detection agent attached. Further, the therapeutic agents donot need to be the same but can be different therapeutic agents, forexample, one can attach a drug and a radioisotope to the same fusionprotein. Particularly, an IgG can be radiolabeled with ¹³¹I and attachedto a drug. The 1311 can be incorporated into the tyrosine of the IgG andthe drug attached to the epsilon amino group of the IgG lysines. Boththerapeutic and diagnostic/detection agents also can be attached toreduced SH groups and to the carbohydrate side chains.

A wide variety of diagnostic and therapeutic reagents can beadministered concurrently or sequentially, or advantageously conjugatedto the antibodies of the invention, for example, drugs, toxins,oligonucleotides, immunomodulators, hormones, hormone antagonists,enzymes, enzyme inhibitors, therapeutic radionuclides, an angiogenesisinhibitor, etc. The therapeutic agents recited here are those agentsthat also are useful for administration separately with the nakedantibody as described above. Therapeutic agents include, for example,chemotherapeutic drugs such as vinca alkaloids, anthracyclines,gemcitabine, epidophyllotoxins, taxanes, antimetabolites, alkylatingagents, antibiotics, SN-38, COX-2 inhibitors, antimitotics,antiangiogenic and apoptotoic agents, particularly doxorubicin,methotrexate, taxol, CPT-11, camptothecans, and others from these andother classes of anticancer agents, and the like. Other useful cancerchemotherapeutic drugs for administering concurrently or sequentially,or for the preparation of immunoconjugates and antibody fusion proteinsinclude nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes,folic acid analogs, COX-2 inhibitors, pyrimidine analogs, purineanalogs, platinum coordination complexes, hormones, and the like.Suitable chemotherapeutic agents are described in REMINGTON'SPHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and inGOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed.(MacMillan Publishing Co. 1985), as well as revised editions of thesepublications. Other suitable chemotherapeutic agents, such asexperimental drugs, are known to those of skill in the art.

In one embodiment, the humanized PAM4 antibodies and fragments thereofof the present invention is conjugated to gemcitabine. In anotherembodiment, gemcitabine is given before, after, or concurrently with anaked or conjugated humanized PAM4 antibody or fragment thereof of thepresent invention. Preferably, the conjugated humanized PAM4 antibody orantibody fragment is conjugated to a radionuclide.

A toxin can be of animal, plant or microbial origin. A toxin, such asPseudomonas exotoxin, may also be complexed to or form the therapeuticagent portion of an immunoconjugate of the PAM4 and hPAM4 antibodies ofthe present invention. Other toxins suitably employed in the preparationof such conjugates or other fusion proteins, include ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, andPseudomonas endotoxin. See, for example, Pastan et al., Cell 47:641(1986), and Goldenberg, CA—A Cancer Journal for Clinicians 44:43 (1994).Additional toxins suitable for use in the present invention are known tothose of skill in the art and are disclosed in U.S. Pat. No. 6,077,499,which is incorporated in its entirety by reference.

An immunomodulator, such as a cytokine, may also be conjugated to, orform the therapeutic agent portion of the PAM4 and hPAM4immunoconjugate, or may be administered with, but unconjugated to, thehumanized or human PAM4 antibody or fragment thereof, or PAM4 fusionprotein or fragment thereof of the present invention. The PAM4 fusionprotein or fragment thereof may comprise one or more antibodies orfragments thereof binding to different antigens. For example, the fusionprotein may bind the PAM4 antigen as well as immunomodulating cells orfactors. Alternatively, subjects can receive a naked PAM4 antibody,fusion protein, or fragment thereof and a separately administeredcytokine, which can be administered before, concurrently or afteradministration of the naked PAM4 antibodies. As used herein, the term“immunomodulator” includes cytokines, stem cell growth factors,lymphotoxins, such as tumor necrosis factor (TNF), and hematopoicticfactors, such as interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3,IL-6, IL-10, IL-12 and IL-18, IL-21), colony stimulating factors (e.g.,granulocyte-colony stimulating factor (G-CSF) and granulocytemacrophage-colony stimulating factor (GM-CSF)), interferons (e.g.,interferons-α, -β and -γ), the stem cell growth factor designated “S1factor,” erythropoietin and thrombopoietin. Examples of suitableimmunomodulator moieties include IL-2, IL-6, IL-10, IL-12, IL-18, IL-21,interferon-γ, TNF-α, and the like.

Alternatively, the antibodies and fragments of the present invention canbe detectably labeled by linking the antibody to an enzyme. When theantibody-enzyme conjugate is incubated in the presence of theappropriate substrate, the enzyme moiety reacts with the substrate toproduce a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric or visual means. Examples of enzymesthat can be used to detectably label antibody include malatedehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeastalcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase,asparaginase, glucose oxidase, α-galactosidase, ribonuclease, urease,catalase, glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

A therapeutic or diagnostic/detection agent can be attached at the hingeregion of a reduced antibody component via disulfide bond formation. Asan alternative, such agents can be attached to the antibody componentusing a heterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the therapeutic ordiagnostic/detection agent can be conjugated via a carbohydrate moietyin the Fc region of the antibody. The carbohydrate group can be used toincrease the loading of the same agent that is bound to a thiol group,or the carbohydrate moiety can be used to bind a different peptide.

In the methods of the invention, the targetable construct may compriseone or more radioactive isotopes useful for detecting diseased tissue.Particularly useful diagnostic radionuclides include, but are notlimited to, ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga,⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ⁹⁴mTc, ⁹⁴Tc, ⁹⁹mTc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,¹⁵⁴⁻¹⁵⁸Gd, ³²P, ¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As,⁷⁵Br, ⁷⁶Br, ^(82m)Rb, ⁸³Sr, or other gamma-, beta-, orpositron-emitters, preferably with a decay energy in the range of 20 to4,000 keV, more preferably in the range of 25 to 4,000 keV, and evenmore preferably in the range of 25 to 1,000 keV, and still morepreferably in the range of 70 to 700 keV. Total decay energies of usefulpositron-emitting radionuclides are preferably <2,000 keV, morepreferably under 1,000 keV, and most preferably <700 keV. Radionuclidesuseful as diagnostic/detection agents utilizing gamma-ray detectioninclude, but are not limited to: ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu, ⁶⁷Ga,⁷⁵Se, ⁹⁷Ru, ^(99m)Tc, ¹¹¹In, ^(114m)In, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁶⁹Yb, ¹⁹⁷Hg,and ²⁰¹Tl. Decay energies of useful gamma-ray emitting radionuclides arepreferably 20-2000 keV, more preferably 60-600 keV, and most preferably100-300 keV.

In the methods of the invention, the targetable construct may compriseone or more radioactive isotopes useful for treating diseased tissue.Particularly useful therapeutic radionuclides include, but are notlimited to ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y,¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy,¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr,⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹Pb.The therapeutic radionuclide preferably has a decay energy in the rangeof 20 to 6,000 keV, preferably in the ranges 60 to 200 keV for an Augeremitter, 100-2,500 keV for a beta emitter, and 4,000-6,000 keV for analpha emitter. Maximum decay energies of useful beta-particle-emittingnuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, andmost preferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies ofuseful alpha-particle-emitting radionuclides are preferably 2,000-10,000keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000keV.

For example, 67Cu, considered one of the more promising radioisotopesfor radioimmunotherapy due to its 61.5 hour half-life and abundantsupply of beta particles and gamma rays, can be conjugated to a PAM4antibody using the chelating agent,p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA). Chase,supra. Alternatively, ⁹⁰Y, which emits an energetic beta particle, canbe coupled to a PAM4 antibody, fusion protein, or fragment thereof,using diethylenetriaminepentaacetic acid (DTPA).

Additional potential radioisotopes include ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br, ¹⁹⁸Au,²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg,²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt,¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe,⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

In another embodiment, a radiosensitizer can be used in combination witha naked or conjugated PAM4 antibody or antibody fragment of the presentinvention. For example, the radiosensitizercan be used in combinationwith a radiolabeled PAM4 antibody or antibody fragment. The addition ofthe radiosensitizer can result in enhanced efficacy when compared totreatment with the radiolabeled antibody or antibody fragment alone.Radiosensitizers are described in D. M. Goldenberg (ed.), CANCER THERAPYWITH RADIOLABELED ANTIBODIES, CRC Press (1995), which is incorporatedherein by reference in its entirety.

The PAM4 antibody or fragment thereof, or PAM4 fusion protein orfragment thereof of the present invention that have a boronaddend-loaded carrier for thermal neutron activation therapy willnormally be effected in similar ways. However, it will be advantageousto wait until non-targeted PAM4 immunoconjugate clears before neutronirradiation is performed. Clearance can be accelerated using an antibodythat binds to the PAM4 antibody. See U.S. Pat. No. 4,624,846 for adescription of this general principle. For example, boron addends suchas carboranes, can be attached to PAM4 antibodies. Carboranes can beprepared with carboxyl functions on pendant side chains, as iswell-known in the art. Attachment of carboranes to a carrier, such asaminodextran, can be achieved by activation of the carboxyl groups ofthe carboranes and condensation with amines on the carrier. Theintermediate conjugate is then conjugated to the PAM4 antibody. Afteradministration of the PAM4 antibody conjugate, a boron addend isactivated by thermal neutron irradiation and converted to radioactiveatoms which decay by α-emission to produce highly toxic, short-rangeeffects.

Furthermore, the present invention includes methods of diagnosing cancerin a subject. Diagnosis may be accomplished by administering adiagnostically effective amount of a diagnostic conjugate, formulated ina pharmaceutically suitable excipient, and detecting said label. ThePAM4 antibodies, fusion proteins, and fragments thereof may beconjugated to the diagnostic/detection agent or be administeredunconjugated to the diagnostic/detection agent, but before,concurrently, or after administration of the diagnostic/detection agent.Radioactive agents that can be used as diagnostic/detection agents werediscussed above. A suitable non-radioactive diagnostic/detection agentis a contrast agent suitable for magnetic resonance imaging, X-rays,computed tomography or ultrasound. Magnetic imaging agents include, forexample, non-radioactive metals, such as manganese, iron and gadolinium,complexed with metal-chelate combinations that include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, when used along with theantibodies of the invention. See U.S. Ser. No. 09/921,290 filed on Oct.10, 2001, which is incorporated in its entirety by reference.

Contrast agents, such as MRI contrast agents, contemplated in thepresent invention include, for example, gadolinium ions, lanthanum ions,dysprosium ions, iron ions, manganese ions or other comparable label, CTcontrast agents, and ultrasound contrast agents are suitable for use inthe present invention.

Paramagnetic ions suitable for the present invention include includechromium (III), manganese (II), iron (III), iron (II), cobalt (II),nickel (II), copper (II), neodymium (III), samarium (III), ytterbium(III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III),holmium (III) and erbium (III), with gadolinium being particularlypreferred.

Ions useful in other contexts, such as X-ray imaging, include but arenot limited to lanthanum (III), gold (III), lead (II), and especiallybismuth (III). Fluorescent labels include rhodamine, fluorescein andrenographin. Rhodamine and fluorescein are often linked via anisothiocyanate intermediate.

Metals are also useful in diagnostic/detection agents, including thosefor magnetic resonance imaging techniques. These metals include, but arenot limited to: Gadolinium, manganese, iron, chromium, copper, cobalt,nickel, dysprosium, rhenium, europium, terbium, holmium and neodymium.In order to load an antibody component with radioactive metals orparamagnetic ions, it may be necessary to react it with a reagent havinga long tail to which are attached a multiplicity of chelating groups forbinding the ions. Such a tail can be a polymer such as a polylysine,polysaccharide, or other derivatized or derivatizable chain havingpendant groups to which can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose. Chelates are coupled to the PAM 4 antibody, fusionprotein, or fragments thereof using standard chemistries. The chelate isnormally linked to the antibody by a group which enables formation of abond to the molecule with minimal loss of immunoreactivity and minimalaggregation and/or internal cross-linking. Other, more unusual, methodsand reagents for conjugating chelates to antibodies are disclosed inU.S. Pat. No. 4,824,659 to Hawthorne, entitled “Antibody Conjugates”,issued Apr. 25, 1989, the disclosure of which is incorporated herein inits entirety by reference. Particularly useful metal-chelatecombinations include 2-benzyl-DTPA and its monomethyl and cyclohexylanalogs, used with diagnostic isotopes in the general energy range of 20to 2,000 keV. The same chelates, when complexed with non-radioactivemetals, such as manganese, iron and gadolinium are useful for MRI, whenused along with the antibodies of the invention. Macrocyclic chelatessuch as NOTA, DOTA, and TETA are of use with a variety of metals andradiometals, most particularly with radionuclides of gallium, yttriumand copper, respectively. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelates such as macrocyclic polyethers, which are of interestfor stably binding nuclides, such as 223Ra for RAIT are encompassed bythe invention.

Radiopaque and contrast materials are used for enhancing X-rays andcomputed tomography, and include iodine compounds, barium compounds,gallium compounds, thallium compounds, etc. Specific compounds includebarium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid,iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide,iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid,ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetricacid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallouschloride.

The antibodies, fusion proteins, and fragments thereof of the presentinvention also can be labeled with a fluorescent compound. The presenceof a fluorescent-labeled MAb is determined by exposing the antibody tolight of the proper wavelength and detecting the resultant fluorescence.Fluorescent labeling compounds include fluorescein isothiocyanate,rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine. Fluorescently-labeled antibodies are particularlyuseful for flow cytometry analysis.

Alternatively, the antibodies, fusion proteins, and fragments thereof ofthis invention can be detectably labeled by coupling the antibody to achemiluminescent compound. The presence of the chemiluminescent-taggedMAb is determined by detecting the presence of luminescence that arisesduring the course of a chemical reaction. Examples of chemiluminescentlabeling compounds include luminol, isoluminol, an aromatic acridiniumester, an imidazole, an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label the antibodiesand fragments thereof the present invention. Bioluminescence is a typeof chemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Bioluminescent compounds that are useful forlabeling include luciferin, luciferase and acquorin.

Accordingly, a method of diagnosing a malignancy in a subject isdescribed, comprising performing an in vitro diagnosis assay on aspecimen (fluid, tissue or cells) from the subject with a compositioncomprising a naked PAM4 MAb or fragment thereof or a naked antibodyfusion protein or fragment thereof. Immunohistochemistry can be used todetect the presence of PAM4 in a cell or tissue. Preferably, themalignancy that is being diagnosed is a cancer. Most preferably, thecancer is pancreatic cancer.

Additionally, a chelator such as DTPA, DOTA, TETA, or NOTA or a suitablepeptide, to which a detectable label, such as a fluorescent molecule, orcytotoxic agent, such as a heavy metal or radionuclide, can beconjugated. For example, a therapeutically useful immunoconjugate can beobtained by conjugating a photoactive agent or dye to an antibody fusionprotein. Fluorescent compositions, such as fluorochrome, and otherchromogens, or dyes, such as porphyrins sensitive to visible light, havebeen used to detect and to treat lesions by directing the suitable lightto the lesion. In therapy, this has been termed photoradiation,phototherapy, or photodynamic therapy (Jori et al. (eds.), PHOTODYNAMICTHERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985); van denBergh, Chem. Britain 22:430 (1986)). Moreover, monoclonal antibodieshave been coupled with photoactivated dyes for achieving phototherapy.Mew et al., J. Immunol. 130:1473 (1983); idem., Cancer Res. 45:4380(1985); Oseroff et al., Proc. Natl. Acad. Sci. USA 83:8744 (1986);idem., Photochem. Photobiol. 46:83 (1987); Hasan et al., Prog. Clin.Biol. Res. 288:471 (1989); Tatsuta et al., Lasers Surg. Med. 9:422(1989); Pelegrin et al., Cancer 67:2529 (1991). However, these earlierstudies did not include use of endoscopic therapy applications,especially with the use of antibody fragments or subfragments. Thus, thepresent invention contemplates the therapeutic use of immunoconjugatescomprising photoactive agents or dyes.

For purposes of therapy, the PAM4 antibodies and fragments thereof ofthe present invention are administered to a patient in a therapeuticallyeffective amount. An antibody is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient.

A diagnostic/detection agent is a molecule or atom, which may beadministered conjugated to an antibody moiety, i.e., antibody orantibody fragment, or subfragment, fusion protein, and fragments thereofand is useful in diagnosing/detecting a disease by locating the cellscontaining the disease-associated antigen. Useful diagnostic/detectionagents include, but are not limited to, radioisotopes, dyes (such aswith the biotin-streptavidin complex), radiopaque materials (e.g.,iodine, barium, gallium, and thallium compounds and the like), contrastagents, fluorescent compounds or molecules and enhancing agents (e.g.,paramagnetic ions) for magnetic resonance imaging (MRI). U.S. Pat. No.6,331,175 describes MRI technique and the preparation of antibodiesconjugated to a MRI enhancing agent and is incorporated in its entiretyby reference. Preferably, the diagnostic/detection agents are selectedfrom the group consisting of radioisotopes for nuclear imaging,endoscopic and intravascular detection, enhancing agents for use inmagnetic resonance imaging or in ultrasonography, radiopaque andcontrast agents for X-rays and computed tomography, and fluorescentcompounds for fluoroscopy, including endoscopic fluoroscopy. Fluorescentand radioactive agents conjugated to antibodies or used in bispecific,pretargeting methods, are particularly useful for endoscopic,intraoperative or intravascular detection of the targeted antigensassociated with diseased tissues or clusters of cells, such as malignanttumors, as disclosed in Goldenberg U.S. Pat. Nos. 5,716,595, 6, 096,289and U.S. application Ser. No. 09/348,818, incorporated herein byreference in their entirety, particularly with gamma-, beta-, andpositron-emitters. Endoscopic applications may be used when there isspread to a structure that allows an endoscope, such as the colon.Radionuclides useful for positron emission tomography include, but arenot limited to: F-18, Mn-51, Mn-52m, Fe-52, Co-55, Cu-62, Cu-64, Ga-6.8,As-72, Br-75, Br-76, Rb-82m, Sr-83, Y-86, Zr-89, Tc-94m, In-110, I-120,and I-124. Total decay energies of useful positron-emittingradionuclides are preferably <2,000 keV, more preferably under 1,000keV, and most preferably <700 keV. Radionuclides useful asdiagnostic/detection agents utilizing gamma-ray detection include, butare not limited to: Cr-51, Co-57, Co-58, Fe-5 g, Cu-67, Ga-67, Se-75,Ru-97, Tc-99m, In-111, In-114m, I-123, I-125, I-131, Yb-169, Hg-197, andTl-201. Decay energies of useful gamma-ray emitting radionuclides arepreferably 20-2000 keV, more preferably 60-600 keV, and most preferably100-300 keV.

In Vitro Diagnosis

The present invention contemplates the use of PAM4 antibodies, includingPAM4 fusion proteins and fragments thereof, to screen biological samplesin vitro for the presence of the PAM4 antigen. In such immunoassays, thePAM4 antibody, fusion protein, or fragment thereof may be utilized inliquid phase or bound to a solid-phase carrier, as described below. In apreferred embodiment, the PAM4 antibody or fragment thereof ishumanized. Also preferred, the PAM4 antibody or fragment thereof isfully human. Still preferred, the PAM4 fusion protein comprises ahumanized or fully human PAM4 antibody.

One example of a screening method for determining whether a biologicalsample contains the PAM4 antigen is the radioimmunoassay (RIA). Forexample, in one form of RIA, the substance under test is mixed with PAM4antigen MAb in the presence of radiolabeled PAM4 antigen. In thismethod, the concentration of the test substance will be inverselyproportional to the amount of labeled PAM4 antigen bound to the MAb anddirectly related to the amount of free, labeled PAM4 antigen. Othersuitable screening methods will be readily apparent to those of skill inthe art.

Alternatively, in vitro assays can be performed in which a PAM4antibody, fusion protein, or fragment thereof is bound to a solid-phasecarrier. For example, MAbs can be attached to a polymer, such asaminodextran, in order to link the MAb to an insoluble support such as apolymer-coated bead, a plate or a tube.

Other suitable in vitro assays will be readily apparent to those ofskill in the art. The specific concentrations of detectably labeled PAM4antibody and PAM4 antigen, the temperature and time of incubation, aswell as other assay conditions may be varied, depending on variousfactors including the concentration of the PAM4 antigen in the sample,the nature of the sample, and the like. The binding activity of a sampleof PAM4 antibody may be determined according to well-known methods.Those skilled in the art will be able to determine operative and optimalassay conditions for each determination by employing routineexperimentation.

Other such steps as washing, stirring, shaking, filtering and the likemay be added to the assays as is customary or necessary for theparticular situation.

The presence of the PAM4 antigen in a biological sample can bedetermined using an enzyme-linked immunosorbent assay (ELISA). In thedirect competitive ELISA, a pure or semipure antigen preparation isbound to a solid support that is insoluble in the fluid or cellularextract being tested and a quantity of detectably labeled solubleantibody is added to permit detection and/or quantitation of the binarycomplex formed between solid-phase antigen and labeled antibody.

In contrast, a “double-determinant” ELISA, also known as a “two-siteELISA” or “sandwich assay,” requires small amounts of antigen and theassay does not require extensive purification of the antigen. Thus, thedouble-determinant ELISA is preferred to the direct competitive ELISAfor the detection of an antigen in a clinical sample. See, for example,the use of the double-determinant ELISA for quantitation of the c-myconcoprotein in biopsy specimens. Field et al., Oncogene 4: 1463 (1989);Spandidos et al., AntiCancer Res. 9: 821 (1989).

In a double-determinant ELISA, a quantity of unlabeled MAb or antibodyfragment (the “capture antibody”) is bound to a solid support, the testsample is brought into contact with the capture antibody, and a quantityof detectably labeled soluble antibody (or antibody fragment) is addedto permit detection and/or quantitation of the ternary complex formedbetween the capture antibody, antigen, and labeled antibody. An antibodyfragment is a portion of an antibody such as F(ab′)₂, F(ab)₂, Fab′, Fab,and the like. In the present context, an antibody fragment is a portionof a PAM4 MAb that binds to an epitope of the PAM4 antigen. The term“antibody fragment” also includes any synthetic or geneticallyengineered protein that acts like an antibody by binding to a specificantigen to form a complex. For example, antibody fragments includeisolated fragments consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, and recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker. Anantibody fusion protein is a recombinantly produced antigen-bindingmolecule in which two or more of the same or different single-chainantibody or antibody fragment segments with the same or differentspecificities are linked. The fusion protein may comprise a singleantibody component, a multivalent or multispecific combination ofdifferent antibody components or multiple copies of the same antibodycomponent. The fusion protein may additionally comprise an antibody oran antibody fragment conjugated to a diagnostic/detection and/or atherapeutic agent. The term PAM4 antibody includes humanized, human andmurine antibodies, antibody fragments thereof, immunoconjugates andfragments thereof and antibody fusion proteins and fragments thereof.

Methods of performing a double-determinant ELISA are well-known. See,for example, Field et al., supra, Spandidos et al., supra, and Moore etal., “Twin-Site ELISAs for fos and myc Oncoproteins Using the AMPAKSystem,” in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 273-281 (TheHumana Press, Inc. 1992).

In the double-determinant ELISA, the soluble antibody or antibodyfragment must bind to a PAM4 epitope that is distinct from the epitoperecognized by the capture antibody. The double-determinant ELISA can beperformed to ascertain whether the PAM4 antigen is present in a biopsysample. Alternatively, the assay can be performed to quantitate theamount of PAM4 antigen that is present in a clinical sample of bodyfluid. The quantitative assay can be performed by including dilutions ofpurified PAM4 antigen.

The PAM4 Mabs, fusion proteins, and fragments thereof of the presentinvention also are suited for the preparation of an assay kit. Such akit may comprise a carrier means that is compartmentalized to receive inclose confinement one or more container means such as vials, tubes andthe like, each of said container means comprising the separate elementsof the immunoassay.

For example, there may be a container means containing the captureantibody immobilized on a solid phase support, and a further containermeans containing detectably labeled antibodies in solution. Furthercontainer means may contain standard solutions comprising serialdilutions of PAM4 antigen. The standard solutions of PAM4 antigen may beused to prepare a standard curve with the concentration of PAM4 antigenplotted on the abscissa and the detection signal on the ordinate. Theresults obtained from a sample containing PAM4 antigen may beinterpolated from such a plot to give the concentration of PAM4 antigenin the biological sample.

PAM4 antibodies, fusion proteins, and fragments thereof of the presentinvention also can be used to detect the presence of the PAM4 antigen intissue sections prepared from a histological specimen. Such in situdetection can be used to determine the presence of the PAM4 antigen andto determine the distribution of the PAM4 antigen in the examinedtissue. In situ detection can be accomplished by applying adetectably-labeled PAM4 antibody to frozen tissue sections. Studiesindicate that the PAM4 antigen is preserved in paraffin-embeddedsections. General techniques of in situ detection are well-known tothose of ordinary skill. See, for example, Ponder, “Cell MarkingTechniques and Their Application,” in MAMMALIAN DEVELOPMENT: A PRACTICALAPPROACH 113-38 Monk (ed.) (IRL Press 1987), and Coligan at pages5.8.1-5.8.8.

PAM4 antibodies, fusion proteins, and fragments thereof can bedetectably labeled with any appropriate marker moiety, for example, aradioisotope, an enzyme, a fluorescent label, a dye, a chromagen, achemiluminescent label, a bioluminescent labels or a paramagnetic label.Methods of making and detecting such detectably-labeled PAM4 antibodiesare well-known to those of ordinary skill in the art, and are describedin more detail below.

The marker moiety can be a radioisotope that is detected by such meansas the use of a gamma counter or a scintillation counter or byautoradiography. In a preferred embodiment, the diagnostic conjugate isa gamma-, beta- or a positron-emitting isotope. A marker moiety in thepresent description refers to a molecule that will generate a signalunder predetermined conditions. Examples of marker moieties includeradioisotopes, enzymes, fluorescent labels, chemiluminescent labels,bioluminescent labels and paramagnetic labels. As used herein, adiagnostic or therapeutic agent is a molecule or atom which isconjugated to an antibody moiety to produce a conjugate which is usefulfor diagnosis and for therapy. Examples of diagnostic or therapeuticagents include drugs, toxins, oligonucleotides, immunomodulators,cytokines, hormones, hormone antagonists, enzymes, enzyme inhibitors,isotopes, other antibodies, chelators, dyes, chromagens, boroncompounds, and marker moieties.

In one embodiment, an oligonucleotide, such as an antisense moleculeinhibiting bcl-2 expression is described in U.S. Pat. No. 5,734,033(Reed), which is incorporated by reference in its entirety, may beconjugated to, or form the therapeutic agent portion of animmunoconjugate or antibody fusion protein of the present invention.Alternatively, the oligonucleotide may be administered concurrently orsequentially with a naked or conjugated PAM4 antibody or antibodyfragment of the present invention. In a preferred embodiment, theoligonucleotides is an antisense oligonucleotide that preferably isdirected against an oncogene or oncogene product of a B-cell malignancy,such as bcl-2.

Those of skill in the art will know of other suitable labels, which canbe employed in accordance with the present invention. The binding ofmarker moieties to PAM4 antibodies can be accomplished using standardtechniques known to the art. Typical methodology in this regard isdescribed by Kennedy et al., Clin. Chim. Acta 70: 1 (1976), Schurs etal., Clin. Chim. Acta 81: 1 (1977), Shih et al., Int'l J. Cancer 46:1101 (1990).

The above-described in vitro and in situ detection methods may be usedto assist in the diagnosis or staging of a pathological condition. Forexample, such methods can be used to detect tumors that express the PAM4antigen such as pancreatic cancer.

In Vivo Diagnosis

The present invention also contemplates the use of PAM4 antibodies forin vivo diagnosis. The method of diagnostic imaging with radiolabeledMAbs is well-known. In the technique of immunoscintigraphy, for example,antibodies are labeled with a gamma-emitting radioisotope and introducedinto a patient. A gamma camera is used to detect the location anddistribution of gamma-emitting radioisotopes. See, for example,Srivastava (ed.), RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING ANDTHERAPY (Plenum Press 1988), Chase, “Medical Applications ofRadioisotopes,” in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition,Gennaro et al. (eds.), pp. 624-652 (Mack Publishing Co., 1990), andBrown, “Clinical Use of Monoclonal Antibodies,” in BIOTECHNOLOGY ANDPHARMACY 227-49, Pezzuto et al. (eds.) (Chapman & Hall 1993).

For diagnostic imaging, radioisotopes may be bound to the PAM4 antibodyeither directly, or indirectly by using an intermediary functionalgroup. Useful intermediary functional groups include chelators such asethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid.For example, see Shih et al., supra, and U.S. Pat. No. 5,057,313.

The radiation dose delivered to the patient is maintained at as low alevel as possible through the choice of isotope for the best combinationof minimum half-life, minimum retention in the body, and minimumquantity of isotope which will permit detection and accuratemeasurement. Examples of radioisotopes that can be bound to PAM4antibody and are appropriate for diagnostic imaging include ^(99m)Tc and¹¹¹In.

The PAM4 antibodies, fusion proteins, and fragments thereof also can belabeled with paramagnetic ions and a variety of radiological contrastagents for purposes of in vivo diagnosis. Contrast agents that areparticularly useful for magnetic resonance imaging comprise gadolinium,manganese, dysprosium, lanthanum, or iron ions. Additional agentsinclude chromium, copper, cobalt, nickel, rhenium, europium, terbium,holmium, or neodymium. PAM4 antibodies and fragments thereof can also beconjugated to ultrasound contrast/enhancing agents. For example, theultrasound contrast agent is a liposome that comprises a humanized PAM4IgG or fragment thereof. Also preferred, the ultrasound contrast agentis a liposome that is gas filled.

In a preferred embodiment, a bispecific antibody can be conjugated to acontrast agent. For example, the bispecific antibody may comprise morethan one image-enhancing agent for use in ultrasound imaging. In apreferred embodiment, the contrast agent is a liposome. Preferably, theliposome comprises a bivalent DTPA-peptide covalently attached to theoutside surface of the liposome. Still preferred, the liposome is gasfilled.

Pharmaceutically Suitable Excipient

Additional pharmaceutical methods may be employed to control theduration of action of a PAM4 antibody in a therapeutic application.Control release preparations can be prepared through the use of polymersto complex or adsorb the PAM4 antibody, fusion protein, and fragmentthereof. For example, biocompatible polymers include matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. Sherwood et al.,Bio/Technology 10: 1446 (1992). The rate of release of a PAM4 antibody,fusion protein, and fragment thereof from such a matrix depends upon themolecular weight of the PAM4 antibody, fusion protein, and fragmentthereof the amount of PAM4 antibody within the matrix, and the size ofdispersed particles. Saltzman et al., Biophys. J. 55: 163 (1989);Sherwood et al., supra. Other solid dosage forms are described in Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The humanized and human PAM4 antibodies and fragments thereof to bedelivered to a subject can consist of the antibody, immunoconjugate,fusion protein, or fragments thereof alone, or can comprise one or morepharmaceutically suitable excipients, one or more additionalingredients, or some combination of these.

The immunoconjugate, naked antibody, and fragments thereof of thepresent invention can be formulated according to known methods toprepare pharmaceutically useful compositions, whereby theimmunoconjugate or naked antibody is combined in a mixture with apharmaceutically suitable excipient. Sterile phosphate-buffered salineis one example of a pharmaceutically suitable excipient. Other suitableexcipients are well-known to those in the art. See, for example, Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The immunoconjugate or naked antibody of the present invention can beformulated for intravenous administration via, for example, bolusinjection or continuous infusion. Formulations for injection can bepresented in unit dosage form, e.g., in ampules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The immunoconjugate, naked antibody, and fragments thereof may also beadministered to a mammal subcutaneously or even by other parenteralroutes. In a preferred embodiment, the PAM4 antibody or fragment thereofis administered in a dosage of 20 to 2000 milligrams protein per dose.Moreover, the administration may be by continuous infusion or by singleor multiple boluses. In general, the dosage of an administeredimmunoconjugate, fusion protein or naked antibody for humans will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of immunoconjugate,antibody fusion protein or naked antibody that is in the range of fromabout 1 mg/kg to 20 mg/kg as a single intravenous infusion, although alower or higher dosage also may be administered as circumstancesdictate. This dosage may be repeated as needed, for example, once perweek for four to ten weeks, preferably once per week for eight weeks,and more preferably, once per week for four weeks. It may also be givenless frequently, such as every other week for several months. The dosagemay be given through various parenteral routes, with appropriateadjustment of the dose and schedule.

The PAM4 antibodies, fusion proteins, and fragments thereof of thepresent invention can be formulated according to known methods toprepare pharmaceutically useful compositions, whereby PAM4 antibodies,fusion proteins and fragments thereof are combined in a mixture with apharmaceutically acceptable carrier. A composition is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well-known to those in the art. See, for example,REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (1990).

For purposes of therapy, the immunoconjugate, or naked antibody isadministered to a mammal in a therapeutically effective amount. Asuitable subject for the present invention are usually a human, althougha non-human animal subject is also contemplated. An antibody preparationis said to be administered in a “therapeutically effective amount” ifthe amount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient mammal.

EXAMPLES

The examples below are illustrative of embodiments of the currentinvention and should not be used, in any way, to limit the scope of theclaims.

The following examples discuss experimental studies employing PAM4 MAband the CaPan1 human pancreatic cancer. The CaPan1 human pancreaticcancer is carried as a xenograft in both subcutaneous and orthotopicsites. The MAb and agent have resulted in significantly improvedsurvival time. High concentrations of PAM4 monoclonal antibody are shownto target xenografted human tumor models to target the majority ofpancreatic tumors within an initial group of patients. Employing an invitro immunoassay to quantitate PAM4-reactive antigen in the blood ofpatients appears promising in its ability to discriminate pancreaticcancer from pancreatitis, as well as other disease and normal groups.

Clinical studies with PAM4 MAb have shown that a majority of the lesionswere targeted in patients and that there is no indication of uptake innormal tissues. Dosimetry indicated that it was possible to deliver 10to 20 cGy/mCi to tumors, with a tumor to red marrow dose ratio of 3:1 to10:1. These data suggest that PAM4 may be useful for development of aphase-I trial for the treatment of pancreatic cancer.

Example 1 Immunohistochemistry Staining Studies

Immunohistochemistry on normal adult tissues showed that the PAM4reactive epitope was restricted to the gastrointestinal tract wherestaining was weak, yet definitely positive (Table 1). Normal pancreatictissue, including ducts, ductules, acini, and islet cells, were negativefor staining. A PAM4 based enzyme immunoassay with tissue homogenates asantigens generally supported the immunohistology data (Table 2). ThePAM4 epitope was absent from normal pancreas and othernongastrointestinal tissues. In neoplastic tissues, PAM4 was reactivewith twenty one out of twenty five (85%) pancreatic cancers (Table 3).PAM4 reactivity appeared to correlate with the stage of tumordifferentiation. For example, twenty out of twenty one well andmoderately differentiated pancreatic tumors were positive whereas onlyone out of four poorly differentiated tumors were positive. Generally,poorly differentiated tumors represent less than 10% of all pancreaticcancers.

These studies have shown the PAM4 reactivity and tissue distribution(both normal and cancer) to be unlike that reported for CA19.9, DUPAN2,SPAN1, Nd2, B72.3, and the Lewis antigens. Together with crossblockingstudies performed with certain of these MAbs, the data suggests that thePAM4 MAb recognizes a unique and novel epitope. When compared to CA19.9,DUPAN2, and aLe^(a), PAM4 appears to be more restricted in its tissuedistribution and it is reactive with a higher percentage of pancreatictumors. Moreover, it gives a greater overall intensity of reaction atequivalent concentrations and is reactive with a higher percentage ofcells within the tumors. Finally, PAM4 was found to be only weaklyreactive with three out of twelve chronic pancreatitis specimens,whereas CA19.9 and DUPAN2 were strongly reactive with all twelvespecimens. Although it is recognized that specificity is dependent uponthe type of assay employed and the range and number of tissues examined,the ability of PAM4 to discriminate between normal and neoplasticpancreatic tissue, its ability to react with a large percentage of thecancer specimens, as well as the high intensity of the reactions, wereimportant rationales for pursuing developmental studies of clinicalapplication. TABLE 1 Immunoperoxidase Staining of Normal Adult Tissueswith MAb PAM4 Staining Tissue Reaction Pancreas (22)^(a) Ducts — Acini —Islets — Submaxillary gland (2) — Esophagus (2) — Stomach (3) +mucussecreting cells Duodenum (3) +goblet cells Jejunum (3) +goblet cellsIleum (3) +goblet cells Colon (5) +goblet cells Liver (3) — Gallbladder(2) — Bronchus (3) — Lung (3) — Heart (3) — Spleen (3) — Kidney (3) —Bladder (3) — Prostate (2) — Testes (2) — Uterus (2) — Ovary (2) —^(a)( ) number of individual specimens examined.

TABLE 2 Monoclonal Antibody PAM4 Reactivity with Normal Adult TissueHomogenates by EIA Tissue ug/g tissue^(a) Pancreas 6.4 Esophagus 8.1Stomach 61.3 Duodenum 44.7 Jejunum 60.6 Colon 74.5 Liver 0.0 Gallbladder5.6 Heart 3.7 Spleen 3.4 Kidney 6.6 Bladder 4.9 Thyroid 3.5 Adrenal 1.3Ureter 2.6 Testes 3.9 CaPan1 Pancreatic Tumor 569^(a)values are mean from two autopsy specimens

TABLE 3 Immunohistochemical Reactivity of Several Monoclonal Antibodieswith Pancreatic Tumors Differentiation PAM4 CA19.9 aLe^(a) DUPAN2  1 W+++ − − +++  2 M ++ +++ +++ +  3 M + − + +  4 M +++ +++ +++ +  5 M ++ +− −  6 M + ND ND ND  7 M* +++ +++ +++ +++  8 M + − − +++  9 M ++ + ++ −10 M* ++ ++ ++ +++ 11 M ++ +++ +++ + 12 M ++ + + +++ 13 M + +++ +++ + 14M ++ + + ++ 15 M +++ + + ++ 16 M + + ++ − 17 M − + + − 18 M ++ ++ ++ ++19 M +++ + +++ ++ 20 M + − − − 21 M +++ +++ + ++ 22 P + + + +++ 23 P − −− − 24 P − − − − 25 P − − + − TOTAL 21/25 17/24 18/24 16/24−: Negative;+: 5-20% of tissue is stained;++: 21-50% of tissue is stained;+++: >50% of tissue is stained;W, M, P: Well, moderate, or poor differentiation;*Metastatic tissue;ND: Not Done

TABLE 4 Immunoperoxidase Staining of Neoplastic Tissues with MAb PAM4Tissue Positive/Total Pancreas 21/25 Colon 10/26 Stomach 1/5 Lung  1/15Breast  0/30 Ovarian  0/10 Prostate 0/4 Liver  0/10 Kidney 0/4

Example 2 In Vivo Biodistribution and Tumor Targeting of RadiolabeledPAM4

Initial biodistribution studies of PAM4 were carried out in a series offour different xenografted human pancreatic tumors covering the range ofexpected differentiation. Each of the four tumor lines employed, AsPc1,BxPc3, Hs766T and CaPan1, exhibited concentrations of ¹³¹I-PAM4 withinthe tumors (range: 21%-48% ID/g on day three) that was significantly(p<0.01-0.001) higher than concomitantly administered nonspecific,isotype-matched Ag8 antibody (range: 3.6%-9.3% ID/g on day three). Thebiodistribution data were used to estimate potential radiation doses tothe tumor of 12,230; 10,684; 6,835; and 15,843 cGy/mCi of injected doseto AsPc1, BxPc3, Hs766T and CaPan1, respectively. With an actual maximumtolerated dose (MTD) of 0.7mCi, PAM4 could provide substantial rad doseto each of the xenografted tumor models. In each tumor line the bloodlevels of radiolabeled PAM4 were significantly (p<0.01-0.001) lower thanthe nonspecific Ag8. Potential radiation doses to the blood from PAM4were 1.4-4.4 fold lower than from Ag8. When radiation doses to the tumorfrom PAM4 were normalized to the blood doses from PAM4, the tumorsreceived doses that were 2.2; 3.3; 3.4; and 13.1-fold higher than blood,respectively. Importantly, potential radiation doses to non-tumortissues were minimal.

The biodistribution of PAM4 was compared with an anti-CEA antibody,MN14, using the CaPan1 tumor model. The concentration of PAM4 within thetumor was much greater than the MN14 at early timepoints, yieldingtumor:blood ratios at day three of 12.7±2.3 for PAM4 compared to 2.7±1.9for MN14. Although PAM4 uptake within the tumor was significantly higherthan for MN14 at early timepoints (day one—p<0.001; day three—p<0.01),dosimetry analyses indicated only a 3.2-fold higher dose to the tumorfrom PAM4 as compared to MN14 over the fourteen day study period. Thiswas due to a rapid clearance of PAM4 from the tumor, such that at latertimepoints similar concentrations of the two antibodies were presentwithin the tumors. A rapid clearance of PAM4 from the tumor was alsonoted in the BxPc3 and Hs766T but not AsPc1 tumor models. Theseobservations were unlike those reported for other anti-mucin antibodies,as for example G9 and B72.3 in colorectal cancer, where each exhibitedlonger retention times as compared to the MN14 antibody. Results fromstudies on the metabolism of PAM4, indicate that after initial bindingto the tumor cell, antibody is rapidly released, possibly beingcatabolized or being shed as an antigen:antibody complex. This mighthave had unfavorable implications for the use of the antibody inpatients except that the blood clearance is also very rapid. These datasuggest that ¹³¹I may not be the appropriate choice of isotope fortherapeutic applications. A short-lived isotope, such as ⁹⁰Y or ¹⁸⁸Re,that can be administered frequently may prove to be a more effectivereagent.

PAM4 showed no evidence of targeting to normal tissues, except in theCaPan1 tumor model, where a small but statistically significant splenicuptake was observed (range 3.1-7.5% ID/g on day three). This type ofsplenic targeting has been observed in the clinical application of theanti-mucin antibodies B72.3 and CC49. Importantly, these studies alsoreported that splenic targeting did not affect tumor uptake of antibodynor did it interfere with interpretation of the nuclear scans. Thesestudies suggested that splenic targeting was not due to crossreactiveantigens in the spleen, nor to binding by Fc receptors, but rather toone or more of the following possibilities: direct targeting of antigentrapped in the spleen, or indirect uptake of antigen:antibody complexesformed either in the blood or released from the tumor site. The latterwould require the presence of immune complexes in the blood; however,these were not observed when specimens as early as five minutes and aslate as seven days were examined by gel filtration (HPLC, GF-250column); radiolabeled antibody eluted as native material. The formerexplanation seems more likely in view of the fact that the CaPan1 tumorproduced large quantities of PAM4-reactive antigen, 100 to 1000-foldhigher than for the other tumor cell lines examined. The lack of splenictargeting by PAM4 in these other tumor lines suggests that thisphenomenon was related to excessive antigen production. In any event,splenic targeting can be overcome by increasing the protein dose to 10ug from the original 2 ug dose. A greater amount of the splenicentrapped antigen presumably was complexed with unlabeled PAM4 ratherthan radiolabeled antibody. Increasing the protein dose had no adverseeffect upon targeting of PAM4 to the tumor or nontumor tissues. In fact,an increase of the protein dose to 100 ug more than doubled theconcentration of radiolabeled PAM4 within the CaPan1 tumor.

Example 3 Development of Orthotopic Pancreatic Tumor Model in AthymicNude Mice

In order to resemble the clinical presentation of pancreatic cancer inan animal model more closely, applicants developed an orthotopic modelby injecting of tumor cells directly into the head of the pancreas.Orthotopic CaPan1 tumors grew progressively without overt symptoms untilthe development of ascites and death at ten to fourteen weeks. By threeto four weeks post-implantation, animals developed a palpable tumor ofapproximately 0.2 g. Within eight weeks of growth, primary tumors ofapproximately 1.2 g along with metastases to the liver and spleen wereobserved (1-3 metastatic tumors/animal; each tumor <0.1 g). At ten tofourteen weeks seeding of the diaphragm with development of ascites wereevident. Ascites formation, and occasional jaundice, were usually thefirst overt indications of tumor growth. Ascites is an accumulation offluid in the abdominal cavity and jaundice is a yellowing of the skinand eyes due to excessive bile pigments in the blood. At this timetumors were quite large, 1 to 2 g, and animals had at most only three tofour weeks until death occurred.

Radiolabeled ¹³¹I-PAM4, administered to animals bearing four week oldorthotopic tumors (approximately 0.2 g) showed specific targeting to theprimary tumor with localization indices of 7.9±3.0 at day one increasingto 22.8±15.3 at day fourteen. No evidence of specific targeting to othertissues was noted. In one case where tumor metastases to the liver andspleen were observed, both metastases were targeted, and had highconcentrations of radiolabeled antibody. In addition, approximately halfof the animals developed a subcutaneous tumor at the incision site. Nosignificant differences were noted in the targeting of orthotopic andsubcutaneous tumors within the same animal, and no significantdifferences were observed in the targeting of orthotopic tumor whetheror not the animal had an additional subcutaneous tumor. The estimatedradiation doses from PAM4 were 6,704 and 1,655 cGy/mCi to the primarytumor and blood, respectively.

Example 4 Development of an Enzyme Immunoassay for Quantification ofCirculating Tumor Antigen

We have developed an enzyme immunoassay employing PAM4 as the capturereagent with an unlabeled, purified IgG derived from rabbit polyclonal,anti-pancreatic mucin, followed by peroxidase labeled donkey anti-rabbitIgG as the detection reagent. The following results were obtainedthrough use of this assay.

Within the range of antigen detected by the assay, coefficient ofvariation values were obtained of less than 10%. Sera from twenty fivehealthy individuals were examined and exhibited a mean±S.D. of 4.0±3.1units. A cutoff value for positive response was then set to the mean±2S.D.=10.2 units. Out of a total of thirty seven pancreatic cancerpatients, thirty two or 86% were positive by this assay, whereas onlythree out of thirteen pancreatitis patients were positive. PAM4 antigenwas elevated in 55% ({fraction (18/33)}) of colorectal cancer patients,a number roughly similar to the 40% of colorectal cancer specimensreactive with PAM4 by immunohistochemistry. Amongst other cancers, PAM4antigen was positive in four out of sixteen ovarian cancer, and five outof twenty breast cancer patients, all of whom had extensive disease.Also, as can be seen in Table 5 below the median value for pancreaticcancer (84.5 units) is on the order of ten fold greater than for all ofthe other cancer groups (except biliary cancer) even though theoverwhelming majority of these cases were late stage, large tumorburden. TABLE 5 PAM4 Reactivity with Sera Units/ml n Mean SD MedianRange % Positive^(a) Normal 25 4.0 3.1 4.7 0.0-9.4  0% Pancreatitis 1314.6 20.3 6.8  0.4-66.7 23% Pancreatic CA 37 317.5 427.1 84.5  0.9-1000 86% Biliary CA  8 155.4 343.8 37.8  6.6-1000  63% Hepatoma CA 30 7.9 8.06.4  0.0-32.8 30% Colorectal CA 33 50.0 171.6 11.8  3.4-1000  55% LungCA 38 25.8 44.6 9.3  0.0-196.0 39% Breast CA 20 11.1 18.5 5.8  0.0-83.325% Ovarian CA 16 68.9 248.4 5.5  0.0-1000  25% Non-Hodgkin's 14 6.6 3.17.5  2.2-12.8 14% Lymphoma^(a)Cutoff 10.2 units/ml (mean + 2 S.D.)

In addition to these findings, a preliminary study was performed in theorthotopic model to examine the potential use of this PAM4 assay inmanagement. At two weeks post-implantation of orthotopic CaPan1 tumor(estimated tumor mass of 0.15 g), none of the animals had detectableantigen in the blood. At four weeks (estimated tumor mass of 0.2 g) oneout of five animals had a detectable level of antigen, (72 units), andat six weeks (estimated tumor volume of 0.4 g) four out of five hadquantifiable antigen (range: 98-6080 units). A severe limiting factor interms of determining the earliest time point at which serum borneantigen could be detected was the limited amount of blood obtainable,such that repeated bleedings could be performed. Thus sera were diluted1:10 prior to assay.

Example 5 Experimental Radioimmunotherapy of Pancreatic Cancer

The initial studies on the use of ¹³¹I-PAM4 for therapy were carried outwith the CaPan1 tumor, which was grown as a subcutaneous xenograft inathymic mice. Animals bearing a 0.25 g tumor were administered 350 μCi,¹³¹I-PAM4 in an experiment that also compared the therapeutic effects ofa similar dose of nonspecific Ag8. The MTD for administration of¹³¹I-PAM4 to animals bearing 1 cm³ tumors is 700 μCi. By weeks five andsix, the PAM4 treated animals showed a dramatic regression of tumor, andeven at week twenty seven, five out of eight remained tumor free. Theuntreated, as well as Ag8-treated animals, showed rapid progression oftumor growth although a significant difference was noted between thesetwo control groups. At seven weeks, tumors from the untreated group hadgrown 20.0±14.6-fold from the initial timepoint whereas the¹³¹I-Ag8-treated tumors had grown only 4.9±1.8-fold. At this time point,the PAM4 tumors had regressed to 0.1±0.1-fold of their original size, asignificant difference from both untreated (p<0.001) and nonspecificAg8-treated (p<0.01) animals.

Although the CaPan1 tumors were sensitive to treatment with ¹³¹I-PAM4,the outcome, that is, regression or progression of the tumor, isdependent upon many factors including initial tumor size. Thus, groupsof animals bearing CaPan1 tumor burdens of 0.25 g, 0.5 g, 1.0 g, or 2.0g were treated with a single dose of the 350 μCi ¹³¹I-PAM4. The majorityof animals having tumors of initial size 0.25 g and 0.5 g (nine of tenanimals in each group) showed tumor regression or growth inhibition forat least sixteen weeks post treatment. In the 1.0 g tumor group five outof seven showed no tumor growth for the sixteen week period and in the2.0 g tumor group six out of nine showed no tumor growth for a period ofsix weeks before progression occurred. Although a single 350 μCi dosewas not as effective against the larger tumors, a single dose may verywell not be the appropriate regimen; toxicity studies indicating theability to give multiple cycles of radioimunotherapy. Animals bearingCaPan1 tumors averaging 1.0 g, were given either a single dose of 350μCi ¹³¹I-PAM4, two doses given at times zero and four weeks or were leftuntreated. The untreated group had a mean survival time of 3.7+/−1.0weeks (survival defined as time for tumor to reach 5 cm³). Animals diedas early as three weeks, with no animal surviving past six weeks. Asingle dose of 350 μCi ¹³¹I-PAM4 produced a significant increase in thesurvival time to 18.8+/−4.2 weeks (p<0.0001). The range of animal deathsextended from weeks thirteen to twenty five. None of the animals werealive at the end of the study period of twenty six weeks.

A significant increase in survival time was observed for the two dosegroup as compared to the single dose group. Half of the animals werealive at the twenty six week timepoint with tumor sizes from 1.0-2.8cm³, and a mean tumor growth rate of 1.6+/−0.7 fold from initial tumorsize. For those animals that were non-survivors at twenty six weeks, themean survival time (17.7+/−5.3 weeks) was similar to the single dosegroup.

Therapy studies with PAM4 have also used the orthotopic tumor model.Groups of animals bearing four week old orthotopic tumors (estimatedtumor weight of 0.25 g) were either left untreated or treated with asingle dose of either 350 uCi ¹³¹I-PAM4 or 350 uCi of ¹³¹I-nonspecificAg8. The untreated animals had a 50% death rate by week ten with nosurvivors at week fifteen. Animals administered nonspecific ¹³¹I-Ag8 atfour weeks of tumor growth, showed a 50% death rate at week seven withno survivors at week fourteen. Although statistically (logrank analysis)there were no differences between these two groups, it is possible thatradiation toxicity had occurred in about half of the Ag8 treatedanimals. Radiolabeled PAM4, however, provided a significant survivaladvantage (p<0.001) as compared to the untreated or Ag8 treated animals,with 70% survival at sixteen weeks, the end of the experiment. At thistime the surviving animals were sacrificed to determine tumor size. Allanimals had tumor with an average weight of 1.2 g, as well as one or twosmall (<0.1 g) metastases evident in four of the seven animals. Atsixteen weeks of growth, these tumors were more representative of aneight week old tumor.

Example 6 Combined Modality Gemzar Chemotherapy and ¹³¹I-PAM4Radioimmunotherapy

Initial studies into the combined use of gemcitabine (gemzar) with¹³¹I-PAM4 radioimmunotherapy were performed as a checkerboard array; asingle dose of Gemzar (0, 100, 200, 500 mg/kg) versus a single dose of¹³¹I-PAM4 ([MTD=700 μCi] 100%, 75%, 50%, 0% of the MTD). The combinedMTD was found to be 500 mg/kg Gemzar with 350 μCi ¹³¹I-PAM4 (50% MTD).Toxicity, as measured by loss of body weight, went to the maximumconsidered as nontoxic; that is 20% loss in body weight. Although thecombined treatment protocol was significantly more effective than gemzaralone, the treatment was no more effective than radioimmunotherapyalone. The next studies were performed at a low dose of gemzar andradioimmunotherapy to examine if a true synergistic therapeutic effectwould be observed. Animals bearing tumors of approximately 1 cm³(approximately 5% of body weight) were administered gemzar, 100 mg/kg ondays zero, three, six, nine, and twelve, with 100 μCi of ¹³¹I-PAM4 givenon day zero. A therapeutic effect was observed with statisticallysignificant (p<0.0001) regression (two of five tumors less than 0.1 cm³)and/or growth inhibition of the tumors compared to gemzar alone. Ofadditional note, in terms of body weight, toxicity was not observed. Thecombination treatment protocol can, if necessary, be delivered inmultiple cycles, with the second treatment cycle beginning in week fouras was done with the radioimmunotherapy alone studies described above.

Example 7 Humanized PAM4 Mab

A preferred embodiment of this invention utilizes the monoclonalantibody, MAb hPAM4, which is a humanized IgG of the murine PAM4 raisedfrom a pancreatic cancer mucin. Humanization of the murine PAM4sequences is utilized to reduce the human antimouse antibody responsethat patients experience. To produce the humanized PAM4, murinecomplementarity determining regions (CDR) are transferred from heavy andlight variable (V) chains of the mouse immunoglobulin into a humanV-domain, followed by the replacement of some human residues in theframework regions with their murine counterparts. Humanized monoclonalantibodies in accordance with this invention are suitable for use in invitro and in vivo diagnostic and therapeutic methods.

Comparison of the variable (V) region framework (FR) sequences of themurine PAM4 MAb (FIGS. 1A and 1B) to registered human antibodies in theKabat database showed that the FRs of PAM4 Vκ and VH exhibited thehighest degree of sequence homology to that of the human antibodiesWalker Vκ and Wil2 VH, respectively. Therefore, the Walker Vκ and Wil2VH FRs were selected as the human frameworks into which the murine CDRsfor PAM4 Vκ and V_(H) were grafted, respectively (FIG. 3). The FR4sequence of the human antibody, NEWM, however, was used to replace theWil2 FR4 sequence for the humanization of the PAM4 heavy chain (FIG.3B). A few amino acid residues in PAM4 FRs that flank the putative CDRswere maintained in hPAM4 based on the consideration that these residueshave more impact on Ag binding than other FR residues. These residuesare 21M, 47W, 59P, 60A, 85S, 87F, and 100G of Vκ and 27Y, 30P, 38K, 48I,66K, 67A, and 69L of VH. The DNA and amino acid sequences of hPAM4 Vκand VH are shown in FIGS. 3A and 3B, respectively.

A modified strategy as described by Leung et al. (Leung et al., 1994))was used to construct the designed Vκ and VH genes for hPAM4 using acombination of long oligonucleotide syntheses and PCR as illustrated inFIG. 4. For the construction of the hPAM4 VH domain, two longoligonucleotides, hPAM4VHA (173-mer) and hPAM4VHB (173-mer) weresynthesized on an automated DNA synthesizer (Applied Biosystem).

hPAM4VHA represents nt 17 to 189 of the hPAM4 VH domain. 5′- AGTCTGGGGCTGAGGTGAAG AAGCCTGGGG CCTCAGTGAA GGTCTCCTGC GAGGCTTCTG GATACACATTCCCTAGCTAT GTTTTGCACT GGGTGAAGCA GGCCCCTGGA CAAGGGCTTG AGTGGATTGGATATATTAAT CCTTACAATG ATGGTACTCA GTACAATGAG AAG-3′

hPAM4VHB represents the minus strand of the hPAM4 VH domaincomplementary to nt 169 to 341. 5′- AGGGTTCCCT GGCCCCAGTA AGCAAATCCGTAGCTACCAC CGAAGCCTCT TGCACAGTAA TACACGGCCG TGTCGTCAGA TCTCAGCCTGCTCAGCTCCA TGTAGGCTGT GTTGATGGAC GTGTCCCTGG TCAGTGTGGC CTTGCCTTTGAACTTCTCAT TGTACTGAGT ACC-3′

The 3′-terminal sequences (21 nt residues) of hPAM4VHA and VHB arecomplementary to each other. Under defined PCR condition, 3′-ends ofhPAM4VHA and VHB anneal to form a short double stranded DNA flanked bythe rest of the long oligonucleotides. Each annealed end serves as aprimer for the transcription of the single stranded DNA, resulting in adouble strand DNA composed of the nt 17 to 341 of hPAM4 VH. This DNA wasfurther amplified in the presence of two short oligonucleotides,hPAM4VHBACK and hPAM4VHFOR to form the fill-length hPAM4 VH. Theunderlined portions are restriction sites for subcloning as shown inFIG. 4B. hPAM4VHBACK 5′-CAG GTG CAG CTG CAG CAG TCT GGG GCT GAG GTG A-3′hPAM4VHFOR 5′-TGA GGA GAC GGT GAC CAG GGT TCC CTG GCC CCA-3′

A minimal amount of hPAM4VHA and VHB (determined empirically) wasamplified in the presence of 10 μL of 10×PCR Buffer (500 mM KCL, 100 mMTris. HCL buffer, pH 8.3, 15 mM MgCl₂), 2 μmol of hPAM4VHBACK andhPAM4VKFOR, and 2.5 units of Taq DNA polymerase (Perkin Elmer Cetus,Norwalk, Conn.). This reaction mixture was subjected to three cycles ofpolymerase chain reaction (PCR) consisting of denaturation at 94° C. for1 minute, annealing at 45° C. for 1 minute, and polymerization at 72° C.for 1.5 minutes. This procedure was followed by 27 cycles of PCRreaction consisting of denaturation at 94° C. for 1 minute, annealing at55° C. for 1 minute, and polymerization at 72° C. for 1 minute.Double-stranded PCR-amplified product for hPAM4 VH was gel-purified,restriction-digested with PstI and BstEII restriction sites and clonedinto the complementary PstI/BstEII restriction sites of the heavy chainstaging vector, VHpBS2, in which the V_(H) sequence was fully assembledwith the DNA sequence encoding the translation initiation codon and asecretion signal peptide in-frame ligated at the 5′-end and an intronsequence at the 3′-end. VHpBS2 is a modified staging vector of VHpBS(Leung et al., Hybridoma, 13:469 (1994)), into which a XhoI restrictionsite was introduced at sixteen bases upstream of the translationinitiation codon to facilitate the next subcloning step. The assembledVH gene was subcloned as a XhoI-BamHI restriction fragment into theexpression vector, pdHL2, which contains the expression cassettes forboth human IgG heavy and light chains under the control of IgH enhancerand MT₁ promoter, as well as a mouse d/fr gene as a marker for selectionand amplification (FIG. 4B). Since the heavy chain region of pdHL2 lacksa BamHI restriction site, this ligation requires use of a linker toprovide a bridge between the BamHI site of the variable chain and theHindIII site present in the pdHL2 vector. The resulting expressionvectors were designated as hPAM4VHpdHL2.

For constructing the full length DNA of the humanized Vκ sequence,hPAM4VKA (157-mer) and hPAM4VKB (156-mer) were synthesized as describedabove. hPAM4VKA and VKB were amplified by two short oligonucleotideshPAM4VKBACK and hPAM4VKFOR as described above.

hPAM4VKA represents nt 16 to 172 of the hPAM4 Vκ domain. 5′-CAGTCTCCATCCTCCCTGTC TGCATCTGTA GGAGACAGAG TCACCATGAC CTGCAGTGCC AGCTCAAGTGTAAGTTCCAG CTACTTGTAC TGGTACCAAC AGAAACCAGG GAAAGCCCCC AAACTCTGGATTTATAGCAC ATCCAACCTG GCTTCTG-3′

hPAM4VKB represents the minus strand of the hPAM4 Vκ domaincomplementary to nt 153 to 308. 5′-GTCCCCCCTC CGAACGTGTA CGGGTACCTATTCCACTGAT GGCAGAAATA AGAGGCAGAA TCTTCAGGTT GCAGACTGCT GATGGTGAGAGTGAAGTCTG TCCCAGATCC ACTGCCACTG AAGCGAGCAG GGACTCCAGA AGCCAGGTTGGATGTG-3′

The 3′-terminal sequences (20 nt residues) of hPAM4VKA and VKB arecomplementary to each other. Under defined PCR condition, 3′-ends ofhPAM4VKA and VKB anneal to form a short double stranded DNA flanked bythe rest of the long oligonucleotides. Each annealed end serves as aprimer for the transcription of the single stranded DNA, resulting in adouble strand DNA composed of the nt 16 to 308 of hPAM4 Vκ. This DNA wasfurther amplified in the presence of two short oligonucleotides,hPAM4VKBACK and hPAM4VKFOR to form the full-length hPAM4 Vκ. Theunderlined portions are restriction sites for subcloning as describedbelow. hPAM4VKBACK 5′-GAC ATC CAG CTG ACC CAG TCT CCA TCC TCC CTG-3′hPAM4VKFOR 5′- TTA GAT CTC CAG TCG TGT CCC CCC TCC GAA CGT-3′

Gel-purified PCR products for hPAM4 Vκ were restriction-digested withPvuII and BglII and cloned into the complementary PvuII/BclI sites ofthe light chain staging vector, VKpBR2. VKpBR2 is a modified stagingvector of VKpBR (Leung et al., Hybridoma, 13:469 (1994)), into which aXbaI restriction site was introduced at sixteen bases upstream of thetranslation initiation codon. The assembled Vκ genes were subcloned asXbaI-BamHI restriction fragments into the expression vector containingthe VH sequence, hPAM4VHpdHL2. The resulting expression vectors weredesignated as hPAM4pdHL2.

Approximately 30 ug of hPAM4pdHL2 was linearized by digestion with SalIand transfected into Sp2/0-Ag14 cells by electroporation at 450 V and 25μF. The transfected cells were plated into 96-well plates and incubatedin a CO₂ cell culture incubator for two days and then selected for MTXresistance. Colonies surviving selection emerged in two to three weeksand were screened for human antibody secretion by ELISA assay. Briefly,supernatants (˜100 ul) from the surviving colonies were added into thewells of an ELISA microplate precoated with goat anti-human IgG F(ab′)₂fragment-specific Ab. The plate was incubated for one hour at roomtemperature. Unbound proteins were removed by washing three times withwash buffer (PBS containing 0.05% Tween-20). Horseradishperoxidase-conjugated goat anti-human IgG Fc fragment-specific Ab wasadded to the wells. Following incubation for one hour, a substratesolution (100 μL/well) containing 4 mM o-phenylenediaminedihydrochloride (OPD) and 0.04% H₂O₂ in PBS was added to the wells afterwashing. Color was allowed to develop in the dark for 30 minutes and thereaction was stopped by the addition of 50 μL of 4 N H₂SO₄ solution. Thebound human IgG was measured by reading the absorbance at 490 nm on anELISA reader. Positive cell clones were expanded and hPAM4 was purifiedfrom cell culture supernatant by affinity chromatography on a Protein Acolumn.

The Ag-binding activity of hPAM4 was confirmed by ELISA assay inmicrotiter plate coated with pancreas cancer cell extractsAn ELISAcompetitive binding assay using PAM4-antigen coated plates weredeveloped to assess the Ag-binding affinity of hPAM4 in comparison withthat of a chimeric PAM4 composed of murine V and human C domains.Constant amounts of the HRP-conjugated cPAM4 mixed with varyingconcentrations of cPAM4 or hPAM4 were added to the coated wells andincubated at room temperature for 1-2 h. The amount of HRP-conjugatedcPAM4 bound to the CaPan1 Ag was revealed by reading the absorbance at490 nm after the addition of a substrate solution containing 4 mMo-phenylenediamine dihydrochloride and 0.04% H₂O₂. As shown by thecompetition assays in FIG. 4, hPAM4 and cPAM4 antibodies exhibitedsimilar binding activities.

Suitable host cells include microbial or mammalian host cells. Apreferred host is the human cell line, PER.C6, which was developed forproduction of MAbs, and other fusion proteins. Accordingly, a preferredembodiment of the present invention is a host cell comprising a DNAsequence encoding a PAM4 MAb, conjugate, fusion protein or fragmentsthereof. PER.C6 cells (WO 97/00326) were generated by transfection ofprimary human embryonic retina cells, using a plasmid that contained theAdserotype 5 (Ad5) E1A- and E1B-coding sequences (Ad5 nucleotides459-3510) under the control of the human phosphoglycerate kinase (PGK)promoter. E1A and E1B are adenovirus early gene activation protein 1Aand 1B, respectively. The methods and compositions are particularlyuseful for generating stable expression of human recombinant proteins ofinterest that are modified post-translationally, e.g. by glycosylation.Several features make PER.C6 particularly useful as a host forrecombinant protein production, such as PER.C6 is a fully characterizedhuman cell line and it was developed in compliance with good laboratorypractices. Moreover, PER.C6 can be grown as a suspension culture indefined serum-free medium devoid of any human- or animal-derivedproteins and its growth is compatible with roller bottles, shakerflasks, spinner flasks and bioreactors with doubling times of about 35hours. Finally, the presence of E1A causes an up regulation ofexpression of genes that are under the control of the CMVenhancer/promoter and the presence of E1B prevents p53-dependentapoptosis possibly enhanced through over expression of the recombinanttransgene. In one embodiment, the cell is capable of producing 2 to200-fold more recombinant protein and/or proteinaceous substance thanconventional mammalian cell lines. Another preferred cell is Sp210-Ag14cell.

Example 8 Therapy of a Patient with Inoperable Pancreatic Carcinoma

A 56-year-old male with extensive, inoperable adenocarcinoma of thepancreas, substantial weight loss (30 lbs of weight or more), lethargyand weakness is given ⁹⁰Y-PAM4 radiolabeled humanized antibody at a doseof 30 mCi of 90-Y and 50 mg antibody protein, in a two hour i.v.infusion. Five days later, the patient is then given a standard courseof gemcitabine chemotherapy. If no evidence after a few months of sideeffects from therapy, the therapy regimen is repeated. During afollow-up examination a few weeks later, it is predicted that thepatient will appear more active and the weight loss will slow. The CTscan of the pancreas is expected to suggest either stable disease or aslight reduction of tumor mass. A repeat examination a few months latershould show, by computed tomography, a substantial reduction of tumormass, and the patient may therefore be considered for resection of thepancreatic tumor mass.

Example 9 Pretargeting with Bispecific PAM4×734 and ^(99m)Tc-or¹¹¹In-Labeled Peptide Haptens

For imaging of pancreatic cancer using a pretargeted approach weprepared a bispecific F(ab′)₂ antibody (bsMAb) consisting of a chimericPAM4 (cPAM4) Fab′ and a murine 734 (m734) Fab′. The m734 antibodyrecognizes an In-DTPA complex. This bsMAb was labeled with ¹²⁵I andinjected (7 Ci; 15 g) into athymic nude mice bearing a human pancreaticcancer xenograft (CaPan1). A non-targeting F(ab′)₂ bsMAb made fromchimeric rituximab (anti-CD20 monoclonal antibody) and m734, was labeledwith ¹³¹I and co-injected as a control. At various time-points (4, 24,36, 48, and 72-hours post-injection) mice were necropsied, the tissuesremoved and counted to determine percent-injected dose per gram (%ID/g). There was significantly greater tumor uptake of bsPAM4 at eachtime-point in comparison to the control bsRituximab (p<0.032 or better).Our past experience with this type of pre-targeting system suggestedthat a blood level of less than 1% ID/g was necessary to obtain goodtumor:non-tumor ratios. At 36-hours post-administration of the bsPAM4there was 1.10±0.40% ID/g in the blood which fell to 0.56±0.08% ID/g at48 hours post-injection. Tumor uptake at these two time-points was6.43±1.50% ID/g and 5.37±2.38% ID/g, respectively. These values weresignificantly higher than the control bsRituximab which had 0.65±0.33%ID/g and 0.47±0.19% ID/g in the tumor at 36 and 48 hours, respectively(p<0.018 and p<0.0098). Blood clearance rates, however, were verysimilar and were not significantly different.

Based on these data, a pre-targeting experiment was carried out inCaPan1 tumor-bearing mice in which radiolabeled peptide-haptens wereinjected 40-hours post-bsMAb administration. Two peptides, IMP-192 andIMP-156, were used, each containing divalent DTPA for recognition by the734 MAb, but one has an additional group specific for binding ^(99m)Tcstably (IMP-192). Tumor-bearing mice (tumor volume ˜0.30 cm³) wereadministered ¹²⁵I-bsPAM4 (6 Ci; 15 g) followed 40 hours later by aradiolabeled peptide-hapten (34.5 Ci; 1.5×10⁻¹¹ moles;bsMAb:peptide=10:1). One group of mice received ^(99m)Tc-labeled IMP192while a second group of mice received ¹¹¹In-labeled IMP156. Controls fornon-specific targeting included two groups that received¹²⁵I-bsRituximab prior to administration of radiolabeled peptide and twoother groups that received ¹¹¹In- or ^(99m)Tc-labeled peptide alone.

Mice were sacrificed at 3 and 24 hours after the administration ofpeptides and the % ID/g determined for the tumor and various tissues.Consistent with our previous findings, there was significantly greaterbsPAM4 in the tumors in comparison to the non-targeting controlbsRituximab, 8.2±3.4% and 0.3±0.08% ID/g, respectively (p<0.0001). Thistranslated into a significantly greatly tumor uptake of ¹¹¹In-IMP156(20.2±5.5% ID/g vs. 0.9+0.1% ID/g, p<0.0001). There was alsosignificantly greater tumor uptake of ^(99m)Tc-IMP192 in the micepre-targeted with bsPAM4 than in those pre-targeted with bsRituximab(16.8±4.8% ID/g vs. 1.1±0.2% ID/g, p<0.0005). Tumor uptake of eachpeptide, when administered alone, was significantly less than in thosemice that received the bsPAM4 (0.2±.05% ID/g and 0.1±0.03% ID/g for^(99m)Tc-IMP 192 and ¹¹¹In-IMP156, p<0.0004 and p<0.0001, respectively).

As with the 3-hour time-point, there was significantly more bsPAM4 inthe tumors at 24 hours post-injection of peptide (64 hours post bsMAbadministration) than bsRituximab (6.4±2.2% ID/g vs. 0.2±0.09% ID/g,respectively; p<0.0001). At this time-point there was 11.1±3.5% ID/g¹¹¹In-IMP156 and 12.9±4.2% ID/g ^(99m)Tc-IMP192 in the tumors of micepre-targeted with bsPAM4 versus 0.5±0.2% ID/g and 0.4±0.03% ID/g inbsRIT pre-targeted tumors (p<0.0008 and p<0.0002, respectively). In themice that received peptide alone, there was significantly less^(99m)Tc-IMP192 in the tumors (0.06±0.02% ID/g, p<0.0007) and¹¹¹In-IMP156 (0.09±0.02% ID/g, p<0.0002) in comparison to the bsPAM4pre-targeted peptides. TABLE 6 Tumor:Non-Tumor Tissue Ratios at EarlyTime- Points. Pre-targeted Pre-targeted ¹²⁵I-bsPAM4 ¹¹¹In-Peptide^(99m)Tc-Peptide F(ab’)₂ (3-Hours) (3-Hours) (4-Hours) Tissue Mean(±STD) Mean (±STD) Mean (±STD) Tumor 1.00 0.00 1.00 0.00 1.00 0.00 Liver36.07 11.74 16.66 7.19 2.34 0.61 Spleen 33.40 20.62 14.62 9.12 2.15 0.74Kidney 7.79 2.81 8.13 3.33 1.10 0.20 Lung 44.55 12.99 15.75 5.85 1.580.37 Blood 36.47 8.28 9.93 5.21 0.47 0.11 Bone 123.24 40.00 — — — — W.Bone 378.00 124.57 — — — — Pancreas 155.55 30.07 73.29 32.85 4.65 1.23Tumor 0.189 (0.070) 0.174 (0.050) 0.179 (0.139) Wt. (g) (±STD)

The table above presents the tumor:non-tumor ratios (T:NT) of varioustissues for these groups, each at an early time-pointpost-administration of radiolabeled product. It is important to notethat at 4-hours post-administration of bsPAM4×m734 F(ab′)₂, thetumor:blood ratio was less than 2:1. However, at 3-hourspost-administration, the pre-targeted ¹¹¹In-IMP156 and ^(99m)Tc-IMP192had significantly greater tumor: nontumor ratios for all tissuesexamined and in particular tumor:blood ratios were equal to 36:1 and9:1, (p<0.001 and p<0.011, respectively). When we examined tumor:bloodratios at the 24-hour time-point, the pre-targeted ¹¹¹In-IMP156 and^(99m)Tc-IMP192 had significantly higher values, 274:1 and 80:1,respectively, versus 4:1 for ¹²⁵I-bsPAM4 alone (p<0.0002). These datastrongly suggest the ability to utilize this pretargeted bsPAM4 approachwith short half-life, high energy radioisotopes that would then deliverhigh radiation dose to tumor with minimal radiation dose to non-tumortissues.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the products, compositions,methods. and processes of this invention. Thus, it is intended that thepresent invention cover such modifications and variations, provided theycome within the scope of the appended claims and their equivalents.

The disclosure of all publications, patents and patent applicationscited above are expressly incorporated herein by reference in theirentireties to the same extent as if each were incorporated by referenceindividually.

1. A humanized antibody or fragment thereof that binds to the domainlocated between the amino terminus and start of the repeat domain of MUC1, wherein said antibody is derived by immunization and/or selectionwith mucin.
 2. The antibody or fragment thereof of claim 1, wherein saidantibody or fragment thereof is raised or selected against mucin ofpancreatic cancer.
 3. The antibody or fragment thereof of claim 1,wherein said antibody or fragment thereof is a PAM4 antibody or fragmentthereof.
 4. The antibody or fragment thereof of claim 3, wherein saidantibody or fragment thereof is a humanized antibody or fragmentthereof.
 5. The antibody or fragment thereof of claim 3, wherein saidantibody or fragment thereof is fully human antibody or fragmentthereof.
 6. A humanized antibody or fragment thereof, comprising thecomplementarity-determining regions (CDRs) of a murine PAM4 MAb and theframework (FR) regions of the light and heavy chain variable regions ofa human antibody and the light and heavy chain constant regions of ahuman antibody, wherein the CDRs of the light chain variable region ofthe humanized PAM4 MAb comprise CDR1 comprising an amino acid sequenceof SASSSVSSSYLY (SEQ ID NO: 1); CDR2 comprising an amino acid sequenceof STSNLAS (SEQ ID NO: 2); and CDR3 comprising an amino acid sequence ofHQWNRYPYT (SEQ ID NO: 3); and the CDRs of the heavy chain variableregion of the humanized PAM4 MAb comprise CDR1 comprising an amino acidsequence of SYVLH (SEQ ID NO: 4); CDR2 comprising an amino acid sequenceof YINPYNDGTQYNEKFKG (SEQ ID NO: 5) and CDR3 comprising an amino acidsequence of GFGGSYGFAY (SEQ ID NO: 6)
 7. The humanized antibody orfragment thereof of claim 4, wherein the FRs of the light and heavychain variable regions of said humanized antibody or fragment thereofcomprise at least one amino acid substituted from the corresponding FRsof a murine PAM4 MAb.
 8. The humanized antibody or fragment thereof ofclaim 7, wherein said substituted amino acid from said murine PAM4 MAbis at least one amino acid selected from the group consisting of aminoacid residue 5, 27, 30, 38, 48, 66, 67, and 69 of the murine heavy chainvariable region of FIG. 1B, PAM4 VH amino acid sequence (SEQ ID NO: 11).9. The humanized antibody or fragment thereof of claim 7, wherein saidamino acid from said murine MAb is at least one amino acid selected fromthe group consisting of amino acid residue 21, 47, 59, 60, 85, 87, and100 of the murine light chain variable region FIG. 1A, PAM4Vκ sequence(SEQ ID NO: 9).
 10. The antibody or fragment thereof of claim 3, whereinsaid antibody or fragment thereof comprises at least one of PAM4 Vκnucleotide sequence (SEQ ID NO: 8) of FIG. 1A and the PAM4 VH nucleotidesequence (SEQ ID NO: 10) of FIG. 1B.
 11. The antibody or fragmentthereof of claim 4, wherein said antibody or fragment thereof comprisesa hPAM4 V_(H) amino acid sequence (SEQ ID NO: 16) of FIG. 4A and a hPAM4Vκ amino acid sequence (SEQ ID NO: 19) of FIG. 4B.
 12. A cancer celltargeting diagnostic or therapeutic conjugate comprising an antibodycomponent that comprises an antibody or fragment thereof of any one ofclaims 1-11, wherein said antibody component is bound to at least onediagnostic and/or therapeutic agent.
 13. The diagnostic conjugateaccording to claim 12, wherein said diagnostic/detection agent isselected from the group comprising a radionuclide, a contrast agent, anda photoactive diagnostic/detection agent.
 14. The diagnostic conjugateof claim 13, wherein said diagnostic/detection agent is a radionuclide.15. The diagnostic conjugate of claim 14, wherein said radionuclide hasan energy between 20 and 4,000 keV.
 16. The diagnostic conjugate ofclaim 14 wherein said radionuclide is a gamma-, beta- or apositron-emitting isotope.
 17. The diagnostic conjugate of claim 16,wherein said radionuclide is selected from the group consisting of¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y,⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd,³²P, ¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br,⁷⁶Br, ^(82m)Rb, ⁸³Sr, or other gamma-, beta-, or positron-emitters. 18.The diagnostic conjugate of claim 13, wherein said diagnostic/detectionagent is a radiological contrast agent.
 19. The diagnostic conjugate ofclaim 18, wherein said contrast agent is a paramagnetic ion.
 20. Thediagnostic conjugate of claim 19, wherein said paramagnetic ion is ametal comprising chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III).
 21. The diagnosticconjugate of claim 18, wherein said contrast agent is a metal comprisinglanthanum (III), gold (III), lead (II), and especially bismuth (III).22. The diagnostic conjugate of claim 18, wherein said contrast agent isan ultrasound enhancing agent.
 23. The diagnostic conjugate of claim 22,wherein said ultrasound enhancing agent is a liposome.
 24. Thediagnostic conjugate of claim 23, wherein said liposome is gas filled.25. The diagnostic conjugate of claim 18, wherein said contrast agent isa radiopaque material selected from the group comprising iodinecompounds, barium compounds, gallium compounds, and thallium compounds.26. The diagnostic conjugate of claim 25, wherein said radiopaquematerial is selected from the group comprising barium, diatrizoate,ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid,iodamide, iodipamide, iodoxamic acid, iogulamide, iohexol, iopamidol,iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamidemeglumine, iosemetic acid, iotasul, iotetric acid, iothalamic acid,iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine,metrizamide, metrizoate, propyliodone, and thallous chloride.
 27. Thediagnostic conjugate of claim 13, wherein said diagnostic/detectionagent is a photoactive diagnostic/detection agent.
 28. The diagnosticconjugate of claim 27, wherein said photoactive diagnostic/detectionagent is a fluorescent labeling compound selected from the groupcomprising fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
 29. Thediagnostic conjugate of claim 27, wherein said photoactivediagnostic/detection agent is a chemiluminescent labeling compoundselected from the group comprising luminol, isoluminol, an aromaticacridinium ester, an imidazole, an acridinium salt and an oxalate ester.30. The diagnostic conjugate of claim 27, wherein said photoactivediagnostic/detection agent is a bioluminescent compound selected fromthe group comprising luciferin, luciferase and aequorin.
 31. Thediagnostic conjugate of claim 13, wherein said conjugate is used inintraoperative, endoscopic, or intravascular tumor diagnosis.
 32. Thetherapeutic conjugate of claim 12, wherein said therapeutic agent isselected from the group consisting of a radionuclide, animmunomodulator, a hormone, a hormone antagonist, an oligonucleotide, anenzyme, an enzyme inhibitor, a photoactive therapeutic agent, acytotoxic agent, an angiogenesis inhibitor, and a combination thereof.33. The therapeutic conjugate of claim 32, wherein said oligonucleotideis an antisense oligonucleotide.
 34. The therapeutic conjugate of claim33, wherein said oligonucleotide is an antisense oligonucleotide againstan oncogene.
 35. The therapeutic conjugate of claim 34, wherein saidoncogene is bcl-2 or p53
 36. The therapeutic conjugate of claim 32,wherein said therapeutic agent is a cytotoxic agent.
 37. The therapeuticconjugate of claim 36, wherein said cytotoxic agent is a drug or atoxin.
 38. The therapeutic conjugate of claim 37, wherein said drugpossesses the pharmaceutical property selected from the group consistingof antimitotic, alkylating, antimetabolite, antiangiogenic, apoptotic,alkaloid, and antibiotic agents and combinations thereof.
 39. Thetherapeutic conjugate of claim 37, wherein said drug is selected fromthe group consisting of nitrogen mustards, gemcitabine, ethyleniminederivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acidanalogs, anthracyclines, taxanes, SN-38, COX-2 inhibitors, pyrimidineanalogs, purine analogs, antibiotics, enzymes, enzyme inhibitors,epipodophyllotoxins, platinum coordination complexes, vinca alkaloids,substituted ureas, methyl hydrazine derivatives, adrenocorticalsuppressants, hormone antagonists, endostatin, taxols, camptothecins,doxorubicins and their analogs, antimetabolites, alkylating agents,antimitotics, antiangiogenic, apoptotoic agents, methotrexate, CPT-11,and a combination thereof.
 40. The therapeutic conjugate of claim 37,wherein said toxin derived from a source selected from the groupcomprising an animal, a plant, and a microbial source.
 41. Thetherapeutic conjugate of claim 37, wherein said toxin is selected fromthe group consisting of ricin, abrin, alpha toxin, saporin, ribonuclease(RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviralprotein, gelonin, diphtherin toxin, Pseudomonas exotoxin, andPseudomonas endotoxin.
 42. The therapeutic conjugate of claim 32,wherein said therapeutic agent is an immunomodulator.
 43. Thetherapeutic conjugate of claim 42, wherein said immunomodulator isselected from the group consisting of a cytokine, a stem cell growthfactor, a lymphotoxin, a hematopoietic factor, a colony stimulatingfactor (CSF), an interferon (IFN), a stem cell growth factor,erythropoietin, thrombopoietin and a combination thereof.
 44. Thetherapeutic conjugate of claim 43, wherein said lymphotoxin is tumornecrosis factor (TNF), said hematopoietic factor is an interleukin (IL),said colony stimulating factor is granulocyte-colony stimulating factor(G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF)),said interferon is interferons-α, -β or -γ, and said stem cell growthfactor is designated “S1 factor”.
 45. The therapeutic conjugate of claim42, wherein said immunomodulator comprises IL-1, IL-2, IL-3, IL-6,IL-10, IL-12, IL-18, IL-21, interferon-γ, TNF-α or a combinationthereof.
 46. The therapeutic conjugate of claim 32, wherein saidtherapeutic agent is a radionuclide.
 47. The therapeutic conjugate ofclaim 46, wherein said radionuclide has an energy between 60 and 700keV.
 48. The therapeutic conjugate of claim 47, wherein saidradionuclide is selected from the group consisting of ³²P, ³³P, ⁴⁷Sc,⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁸⁶Y, ⁹⁰Y, ¹¹¹Ag, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm,¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²¹²Bi, ²¹³Bi,²¹¹At, ²²³Ra and ²²⁵Ac, and combinations thereof.
 49. The therapeuticconjugate of claim 32, wherein said therapeutic agent is a photoactivetherapeutic agent.
 50. The therapeutic conjugate of claim 49, whereinsaid photoactive therapeutic agent is selected from the group comprisingchromogens and dyes.
 51. The therapeutic conjugate of claim 32, whereinsaid therapeutic agent is an enzyme.
 52. The therapeutic conjugate ofclaim 51, wherein said enzyme is selected from the group comprisingmalate dehydrogenase, staphylococcal nuclease, delta-V-steroidisomerase, yeast alcohol dehydrogenase, α-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase.
 53. A multivalent, multispecificantibody or fragment thereof comprising more than one antigen bindingsite having an affinity toward a PAM4 target antigen and one or morehapten binding sites having affinity towards hapten molecules.
 54. Theantibody or fragment thereof of claim 53, wherein said antibody orfragment thereof is a humanized antibody or fragment thereof.
 55. Theantibody or fragment thereof of claim 53, wherein said antibody orfragment thereof is a fully human antibody or fragment thereof.
 56. Theantibody or fragment thereof of claim 53, further comprising adiagnostic or therapeutic agent.
 57. An antibody fusion protein orfragment thereof comprising at least two PAM4 MAbs or fragments thereof.58. An antibody fusion protein or fragment thereof comprising at leastone first PAM4 MAb or fragment thereof of and at least one second MAb orfragment thereof, wherein said second MAb or fragment thereof is not aPAM4 MAb or fragment thereof.
 59. The antibody fusion protein orfragment thereof of claim 58, wherein said second MAb is acarcinoma-associated antibody.
 60. The antibody fusion protein orfragment thereof of claim 59, wherein said carcinoma-associated antibodybinds to an antigen on or is derived from a pancreatic cancer.
 61. Theantibody fusion protein or fragment thereof of claim 59, wherein saidcarcinoma-associated antibody is selected from the group consisting ofCA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, CEA, aLe^(a), antibodiesdefined by the Lewis antigen Le(y), CSAp, MUC1, MUC2, MUC3, MUC4,TAG-72, EGFR, insulin-like growth factor (IGF), tenascin, plateletderived growth factor, IL-6, CD40, angiogenesis factors (e.g., VEGF),products of oncogenes and HER2/neu.
 62. The antibody fusion protein ofclaim 57, wherein said fusion protein further comprises at least onediagnositic or therapeutic agent.
 63. A DNA sequence comprising anucleic acid encoding a MAb or fragment thereof selected from the groupconsisting of (a) a PAM4 antibody or fragment thereof; (b) an antibodyfusion protein or fragment thereof comprising at least two of said PAM 4MAbs or fragments thereof; (c) an antibody fusion protein or fragmentthereof comprising at least one first PAM4 MAb or fragment thereofcomprising said MAb or fragment thereof of and at least one second MAbor fragment thereof, wherein said second MAb or fragment thereof is nota PAM4 MAb or fragment thereof; and (d) an antibody fusion protein orfragment thereof comprising at least one first PAM4 MAb or fragmentthereof and at least one second MAb or fragment thereof, wherein saidsecond MAb is a carcinoma associated antibody.
 64. The DNA sequence ofclaim 63, wherein said carcinoma associated antibody is selected fromthe group consisting of CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, CEA,aLe^(a), antibodies defined by the Lewis antigen Le(y), CD40,angiogenesis factors (e.g., VEGF), products of oncogenes, MUC1, MUC-2,MUC-3, MUC-4, TAG-72, EGFR, insulin-like growth factor (IGF), tenascin,platelet derived growth factor, IL-6, and HER2/neu.
 65. An expressionvector comprising the DNA sequence of claim
 60. 66. A host cellcomprising the DNA sequence of claim
 63. 67. A method of delivering adiagnostic or therapeutic agent, or a combination thereof, to a targetcomprising (i) providing a composition that comprises a PAM4 antibody orfragment thereof conjugated to at least one diagnostic and/ortherapeutic agent and (ii) administering to a subject in need thereofthe diagnostic or therapeutic conjugate of claim
 12. 68. The method ofclaim 67, wherein said diagnostic/detection agent is selected from thegroup consisting of a radionuclide, a contrast agent, and a photoactivediagnostic/detection agent.
 69. The method of claim 68, wherein saiddiagnostic/detection agent is a radionuclide.
 70. The method of claim69, wherein said radionuclide has an energy between 20 and 4,000 keV.71. The method of claim 70, wherein said radionuclide is a gamma-, beta-or a positron-emitting isotope.
 72. The method of claim 71, wherein saidradionuclide is selected from the group consisting of ¹¹⁰In, ¹¹¹In,¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr,^(94m)Tc, ⁹⁴Tc, ^(99m)Tc ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P,¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br,^(82m)Rb, ⁸³ Sr, or other gamma-, beta-, or positron-emitters.
 73. Themethod of claim 68, wherein said diagnostic/detection agent is acontrast agent.
 74. The method of claim 73, wherein said contrast agentis a paramagnetic ion.
 75. The method of claim 74, wherein saidparamagnetic ion is a metal comprising chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) and erbium (III).
 76. Themethod of claim 73, wherein said contrast agent is a metal comprisinglanthanum (III), gold (III), lead (II), and especially bismuth (III).77. The method of claim 73, wherein said contrast agent is an ultrasoundenhancing agent.
 78. The method of claim 77, wherein said ultrasoundenhancing agent is a liposome.
 79. The method of claim 78, wherein saidliposome is gas filled.
 80. The method of claim 73, wherein saidcontrast agent is a radiopaque material selected from the groupcomprising iodine compounds, barium compounds, gallium compounds, andthallium compounds.
 81. The method of claim 80, wherein said radiopaquematerial is selected from the group comprising barium, diatrizoate,ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid,iodamide, iodipamide, iodoxamic acid, iogulamide, iohexol, iopamidol,iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamidemeglumine, iosemetic acid, iotasul, iotetric acid, iothalamic acid,iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine,metrizamide, metrizoate, propyliodone, and thallous chloride.
 82. Themethod of claim 68, wherein said diagnostic/detection agent is aphotoactive diagnostic/detection agent.
 83. The method of claim 82,wherein said photoactive diagnostic/detection agent is a fluorescentlabeling compound selected from the group comprising fluoresceinisothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine.
 84. The method of claim 82, whereinsaid photoactive diagnostic/detection agent is a chemiluminescentlabeling compound selected from the group comprising luminol,isoluminol, an aromatic acridinium ester, an imidazole, an acridiniumsalt and an oxalate ester.
 85. The method of claim 82, wherein saidphotoactive diagnostic/detection agent is a bioluminescent compoundselected from the group comprising luciferin, luciferase and aequorin.86. The method of claim 67, wherein said therapeutic agent is selectedfrom the group consisting of a cytotoxic agent, cytokine,immunomodulator, hormone, a hormone antagonist, growth factor,radionuclide, metal, contrast agent, oligonucleotide, enzyme, enzymeinhibitor, and photoactive therapeutic agent.
 87. The therapeuticconjugate of claim 86, wherein said oligonucleotide is an antisenseoligonucleotide.
 88. The oligonucleotide of claim 87, wherein saidantisense oligonucleotide is an antisense oligonucleotide against anoncogene.
 89. The antisense oligonucleotide of claim 88, wherein saidoncogene is bcl-2 or p53
 90. The method of claim 86, wherein saidtherapeutic agent is a cytoxtoxic agent.
 91. The method of claim 90,wherein said cytotoxic agent is a drug or a toxin.
 92. The method ofclaim 91, wherein said drug possesses the pharmaceutical propertyselected from the group consisting of antimitotic, alkylating,antimetabolite, antiangiogenic, apoptotic, alkaloid, and antibioticagents and combinations thereof.
 93. The method of claim 91, whereinsaid drug is selected from the group consisting of nitrogen mustards,gemcitabine, ethylenimine derivatives, alkyl sulfonates, nitrosoureas,triazenes, folic acid analogs, anthracyclines, SN-38, taxanes, COX-2inhibitors, pyrimidine analogs, purine analogs, antibiotics, enzymes,enzyme inhibitors, epipodophyllotoxins, platinum coordination complexes,vinca alkaloids, substituted ureas, methyl hydrazine derivatives,adrenocortical suppressants, hormone antagonists, endostatin, taxols,camptothecins, doxorubicins and their analogs, antimetabolites,alkylating agents, antimitotics, antiangiogenic, apoptotoic agents,methotrexate, CPT-11, and a combination thereof.
 94. The method of claim91, wherein said toxin derived from a source selected from the groupcomprising an animal, a plant, and a microbial source.
 95. The method ofclaim 94, wherein said toxin is selected from the group consisting ofricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. 96.The method of claim 86, wherein said therapeutic agent is animmunomodulator.
 97. The method of claim 96, wherein saidimmunomodulator is selected from the group consisting of a cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), a stem cell growthfactor, erythropoietin, thrombopoietin and a combination thereof. 98.The method of claim 97, wherein said lymphotoxin is tumor necrosisfactor (TNF), said hematopoietic factor is an interleukin (IL), saidcolony stimulating factor is granulocyte-colony stimulating factor(G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF)),said interferon is interferons-α, -β or -γ, and said stem cell growthfactor is designated “S1 factor”.
 99. The method of claim 96, whereinsaid immunomodulator is a cytokine.
 100. The method of claim 96, whereinsaid immunomodulator comprises IL-1, IL-2, IL-3, IL-6, IL-10, IL-12,IL-18, IL-21, interferon-α, β, or γ, TNF-α or a combination thereof.101. The method of claim 86, wherein said therapeutic agent is aradionuclide.
 102. The method of claim 101, wherein said radionuclidehas an energy between 60 and 700 keV.
 103. The method of claim 96,wherein said radionuclide is selected from the group consisting of ³²P,³³P, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁸⁶Y, ⁹⁰Y, ¹¹¹Ag, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁴²Pr,¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²¹²Bi,²¹³Bi, ²¹¹At, ²²³Ra and ²²⁵Ac, and combinations thereof.
 104. The methodof claim 86, wherein said therapeutic agent is a photoactive therapeuticagent.
 105. The method of claim 104, wherein said photoactivetherapeutic agent is selected from the group comprising chromogens anddyes.
 106. The method of claim 86, wherein said therapeutic agent is anenzyme.
 107. The method of claim 106, wherein said enzyme is selectedfrom the group comprising malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,α-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, β-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.
 108. A method of delivering a diagnostic/detectionagent, a therapeutic agent, or a combination thereof to a target,comprising: (a) administering to a subject the antibody or fragmentthereof of claim 53; (b) waiting a sufficient amount of time for anamount of the non-antibody to clear the subject's blood stream; and (c)administering to said subject a carrier molecule comprising adiagnostic/detection agent, a therapeutic agent, or a combinationthereof, that binds to a binding site of said antibody.
 109. The methodof claim 108, wherein said carrier molecule binds to more than onebinding site of said antibody.
 110. The method of claim 108, whereinsaid diagnostic/detection agent or said therapeutic agent is selectedfrom the group comprising isotopes, drugs, toxins, cytokines, hormones,hormone antagonists, oligonucleotides, enzymes, enzyme inhibitors,growth factors, radionuclides, and metals.
 111. The therapeuticconjugate of claim 110, wherein said oligonucleotide is an antisenseoligonucleotide.
 112. The oligonucleotide of claim 111, wherein saidantisense oligonucleotide is an antisense oligonucleotide against anoncogene.
 113. The antisense oligonucleotide of claim 112, wherein saidoncogene is bcl-2 or p53
 114. A method for diagnosing or treatingcancer, comprising: (a) administering to a subject in need thereof theantibody or fragment thereof of claim 50; (b) waiting a sufficientamount of time for an amount of the non-antibody to clear the subject'sblood stream; and (c) administering to said subject a carrier moleculecomprising a diagnostic/detection agent, a therapeutic agent, or acombination thereof, that binds to a binding site of said antibody. 115.The method of claim 114, wherein said cancer is pancreatic cancer. 116.The method of claim 114, wherein said method can be used forintraoperative identification of diseased tissues, endoscopicidentification of diseased tissues, or intravascular identification ofdiseased tissues.
 117. A method of treating a malignancy in a subjectcomprising: (a) administering to said subject a therapeuticallyeffective amount of an antibody or fragment thereof comprising a PAM4MAb or fragment thereof or PAM4 antibody fusion protein or fragmentthereof, wherein said PAM4 MAb or fragment thereof or PAM4 antibodyfusion protein or fragment thereof is conjugated to at least onetherapeutic agent, and (b) formulating said PAM4 MAb or fragment thereofor antibody fusion protein or fragment thereof in a pharmaceuticallysuitable excipient
 118. The method of claim 117, further comprising asecond MAb or fragment thereof.
 119. The method of claim 118, whereinsaid second MAb or fragment thereof is a naked MAb or fragment thereof.120. The method of claim 118, wherein said second MAb or fragmentthereof is selected from the group consisting of CA19.9, DUPAN2, SPAN1,Nd2, B72.3, CC49, CEA, aLe^(a), antibodies defined by the Lewis antigenLe(y), CSAp, MUC1, MUC-2, MUC-3, MUC-4, TAG-72, EGFR, insulin-likegrowth factor (IGF), tenascin, platelet derived growth factor, IL-6,CD40, angiogenesis factors (e.g., VEGF), products of oncogenes andHER2/neu.
 121. The method of claim 118, wherein said second MAb orfragment thereof is conjugated to a therapeutic or diagnostic/detectionagent.
 122. The method of claim 117, further comprising a second MAb orfragment thereof of any one of claims 1-53.
 123. The method of claim117, wherein said PAM4 antibody is administered parenterally.
 124. Themethod of claim 123, wherein said PAM4 antibody is administered in adosage of 20 to 2000 milligrams protein per dose.
 125. The method ofclaim 124, wherein said dosage is repeatedly administered.
 126. Themethod of claim 117, wherein said PAM4 antibody or fragment thereof isselected from the group consisting of a subhuman primate PAM4 antibodyor fragment thereof, human PAM4 antibody or fragment thereof, andhumanized PAM4 antibody or fragment thereof.
 127. The method of claim126, wherein said human and humanized PAM4 antibody constant and hingeregions comprise constant and hinge regions of a human IgG.
 128. Themethod of claim 117, wherein said PAM4 antibody is administered before,in conjunction with, or after a second naked or conjugated antibodyreactive with a second tumor marker expressed by said malignancy isadministered to said subject.
 129. The method of claim 117, wherein saidPAM4 antibody is administered before, concurrently, or after at leastone therapeutic is administed to said subject.
 130. A method ofdiagnosing a malignancy in a subject comprising (a) administering tosaid subject a diagnostically effective amount of a diagnostic conjugatecomprising a PAM4 MAb or fragment thereof or PAM4 antibody fusionprotein or fragment thereof, wherein said PAM4 MAb or fragment thereofor PAM4 antibody fusion protein or fragment thereof is conjugated to atleast one diagnostic/detection agent, and (b) optionally formulatingsaid PAM4 MAb or fragment thereof or antibody fusion protein or fragmentthereof in a pharmaceutically suitable excipient.
 131. A method oftreating a cancer cell in a subject comprising (i) administering to saidsubject a therapeutically effective amount of a composition comprising anaked PAM4 MAb or fragment thereof or a naked antibody fusion protein orfragment thereof (ii) formulating said naked PAM4 MAb or fragmentthereof or antibody fusion protein or fragment thereof in apharmaceutically suitable excipient.
 132. The method of claim 131,wherein said composition further comprises a second naked antibody orfragment thereof.
 133. The method of claim 132, wherein said secondnaked antibody or fragment thereof is a PAM4 MAb or fragment thereof.134. The method of claim 132, wherein said second antibody or fragmentthereof is not a PAM4 MAb or fragment thereof.
 135. The method of claims132, wherein said second antibody or fragment thereof is selected fromthe group consisting of CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, CEA,aLe^(a), antibodies defined by the Lewis antigen Le(y), CSAp, MUC1,MUC-2, MUC-3, MUC-4, TAG-72, EGFR, insulin-like growth factor (IGF),tenascin, platelet derived growth factor, IL-6, CD40, angiogenesisfactors (e.g., VEGF), products of oncogenes and HER2/neu.
 136. Themethod of claim 134, wherein said second antibody or fragment thereof isselected from the group consisting of CA19.9, DUPAN2, SPAN1, Nd2, B72.3,CC49, CEA, aLe^(a), antibodies defined by the Lewis antigen Le(y), CSAp,MUC 1, MUC-2, MUC-3, MUC-4, TAG-72, EGFR, insulin-like growth factor(IGF), tenascin, platelet derived growth factor, IL-6, CD40,angiogenesis factors (e.g., VEGF), products of oncogenes and HER2/neu.137. The method of claim 131, wherein said naked PAM4 antibody isadministered parenterally.
 138. The method of claim 137, wherein saidnaked PAM4 antibody is administered in a dosage of 20 to 2000 milligramsprotein per dose.
 139. The method of claim 138, wherein said dosage isrepeatedly administered.
 140. The method of claim 131, wherein saidnaked PAM4 antibody is selected from the group consisting of subhumanprimate PAM4 antibody, human PAM4 antibody, and humanized PAM4 antibody.141. The method of claim 140, wherein said human and humanized nakedPAM4 antibody constant and hinge regions comprise constant and hingeregions of a human IgG.
 142. The method of claim 132, wherein said nakedPAM4 antibody or fragment thereof is administered before, in conjunctionwith, or after said second naked antibody is administered to saidsubject.
 143. A method of diagnosing a malignancy in a subjectcomprising (i) performing an in vitro diagnosis assay on a specimen fromsaid subject with a composition comprising a naked PAM4 MAb or fragmentthereof or a naked antibody fusion protein or fragment thereof ofclaim
 1. 144. The method of claim 143, wherein said malignancy is acarcinoma.
 145. The method of claim 144, wherein said cancer ispancreatic cancer.
 146. The method of claim 143, wherein said in vitrodiagnosis assay is selected from the group consisting of immunoassays,PCR, RT-PCR and immunohistochemistry.
 147. The method of claim 146,wherein said in vitro diagnosis assay is RT-PCR or immunoassays. 148.The method of claim 147, wherein said specimen is body fluid or atissue.
 149. The method of claim 146, wherein said diagnosis assay isimmunohistochemistry.
 150. The method of claim 149, wherein saidspecimen is a population of cells or a tissue.
 151. A method ofintraoperatively identifying diseased tissues expressing PAM4 antigen,in a subject, comprising: (A) administering an effective amount of abispecific antibody or antibody fragment comprising at least one armthat specifically binds a targeted tissue expressing PAM4-antigen and atleast one other arm that specifically binds a targetable conjugate,wherein said one arm that specifically binds a targeted tissue is ahPAM4 antibody or fragment thereof; and (B) administering a targetableconjugate selected from the group consisting of (i)DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂; (SEQ ID NO: 7) (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;


152. A method for the endoscopic identification of diseased tissuesexpressing PAM4 antigen, in a subject, comprising: (A) administering aneffective amount of a bispecific antibody or antibody fragmentcomprising at least one arm that specifically binds a targeted tissueexpressing PAM4-antigen and at least one other arm that specificallybinds a targetable conjugate wherein said one arm that specificallybinds a targeted tissue is a hPAM4 antibody or fragment thereof; and (B)administering a targetable conjugate selected from the group consistingof (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂; (SEQ ID NO: 7) (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;


153. A method for the intravascular identification of diseased tissuesexpressing PAM4 antigen, in a subject, comprising: (A) administering aneffective amount of a bispecific antibody or antibody fragmentcomprising at least one arm that specifically binds a targeted tissueexpressing PAM4-antigen and at least one other arm that specificallybinds a targetable conjugate wherein said one arm that specificallybinds a targeted tissue is a hPAM4 antibody or fragment thereof; and (B)administering a targetable conjugate selected from the group consistingof (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂; (SEQ ID NO: 7) (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;


154. A method of detection of lesions during an endoscopic,intravascular catheter, or surgical procedure, wherein the methodcomprises: (a) injecting a subject who is to undergo such a procedurewith a bispecific antibody F(ab)₂ or F(ab′)₂ fragment thereof, diabody,triabody, or tetrabody., wherein said bispecific antibody or fragmentthereof, diabody, triabody or tetrabody has a first antibody bindingsite which specifically binds to a PAM4 antigen, and has a secondantibody binding site which specifically binds to a hapten, andpermitting the antibody fragment to accrete at target sites; (b)optionally clearing non-targeted antibody fragments using agalactosylated anti-idiotype clearing agent if the bispecific fragmentis not largely cleared from circulation within about 24 hours ofinjection, and injecting a bivalent labeled hapten, which quicklylocalizes at the target site and clears through the kidneys; (c)detecting the presence of the hapten by close-range detection ofelevated levels of accreted label at the target sites with detectionmeans, within 48 hours of the first injection, and conducting saidprocedure, wherein said detection is performed without the use of acontrast agent or subtraction agent.
 155. A method for close-rangelesion detection, during an operative, intravascular, or endoscopicprocedure, wherein the method comprises: (a) injecting a subject to sucha procedure parenterally with an effective amount of a hPAM4immunoconjugate or fragment thereof, (b) conducting the procedure within48 hours of the injection; (c) scanning the accessed interior of thesubject at close range with a detection means for detecting the presenceof said labeled antibody or fragment thereof; and (d) locating the sitesof accretion of said labeled antibody or fragment thereof by detectingelevated levels of said labeled antibody or fragment thereof at suchsites with the detection means.